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Computational Study of Structure and Mechanism of Trypanosoma cruzi Trans-Sialidase

Permanent Link: http://ufdc.ufl.edu/UFE0022549/00001

Material Information

Title: Computational Study of Structure and Mechanism of Trypanosoma cruzi Trans-Sialidase
Physical Description: 1 online resource (164 p.)
Language: english
Creator: Demir, Ozlem
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: chagas, computational, cruzi, mechanism, qmmm, rangeli, sialidase, transsialidase, trypanosoma
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Trans-sialidase is a vital enzyme for the lifecycle of Trypanosoma cruzi, the protozoa responsible for Chagas' disease, which is lethal and drastically affects large human populations in Central and South America, widening its epidemic area to North America in recent years. T. cruzi trans-sialidase (TcTS) catalyzes transfer of sialic acids from host glycoconjugates to the parasite's glycoconjugates, which facilitates the parasite the means to escape from the host immune system and to invade the host cells. Thus, TcTS stands as a potential and appealing therapeutic target for Chagas' disease. Experimental evidence suggests that a relatively long-lived covalent intermediate forms in the mechanism of TcTS. If this scenario is correct, sialic acid is scavenged from the host's glycoconjugates and stays bound to the enzyme until the parasite's glycoconjugate enters the active site. However, it is unclear whether the covalent intermediate formation occurs through an SN1 or SN2 mechanism. It is crucial to elucidate the mechanism and the transition structure for future inhibitor design studies of TcTS. Additionally, the common inhibitors for sialidases, which catalyze hydrolysis of sialic acids, do not work for TcTS. The reason for this is unclear since both enzyme families share the first step of the mechanism. Trypanosoma rangeli sialidase (TrSA) stands out among sialidases to perform a comparative study with TcTS due to their distinct structural similarity (%70 sequence identity and C-alpha RMSD of 0.59 ?) and yet, different catalytic function. There is experimental evidence about formation of a covalent intermediate in TrSA as well, but only for an activated ligand. Thus, there is a possibility that the mechanism of TrSA is artificially biased towards covalent intermediate formation due to the effect of substituents on the natural ligand. Elucidating the difference in mechanisms of TcTS and TrSA could also pave the way to tailor sialidases into trans-sialidases (and glycosidases into trans-glycosidases) to use for efficient synthesis of molecules that currently require long and low-yield chemical processes. In this study, the mechanisms of both enzymes are investigated using two different QM/MM methods in Chapters 3 and 4. Potential energy surfaces are constructed for each enzyme by performing constrained minimizations. Based on the potential energy surfaces, the difference in the mechanisms of the two enzymes is discussed. Furthermore, 50-ns long molecular dynamics simulations are performed for the two enzymes in free, ligand-bound and inhibitor-bound forms and these simulations are analyzed thoroughly in Chapter 5 to distinguish any structural or dynamical differences between the two enzymes and to shed light on the reason of difference in their inhibitor binding ability.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Ozlem Demir.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Roitberg, Adrian E.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022549:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022549/00001

Material Information

Title: Computational Study of Structure and Mechanism of Trypanosoma cruzi Trans-Sialidase
Physical Description: 1 online resource (164 p.)
Language: english
Creator: Demir, Ozlem
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: chagas, computational, cruzi, mechanism, qmmm, rangeli, sialidase, transsialidase, trypanosoma
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Trans-sialidase is a vital enzyme for the lifecycle of Trypanosoma cruzi, the protozoa responsible for Chagas' disease, which is lethal and drastically affects large human populations in Central and South America, widening its epidemic area to North America in recent years. T. cruzi trans-sialidase (TcTS) catalyzes transfer of sialic acids from host glycoconjugates to the parasite's glycoconjugates, which facilitates the parasite the means to escape from the host immune system and to invade the host cells. Thus, TcTS stands as a potential and appealing therapeutic target for Chagas' disease. Experimental evidence suggests that a relatively long-lived covalent intermediate forms in the mechanism of TcTS. If this scenario is correct, sialic acid is scavenged from the host's glycoconjugates and stays bound to the enzyme until the parasite's glycoconjugate enters the active site. However, it is unclear whether the covalent intermediate formation occurs through an SN1 or SN2 mechanism. It is crucial to elucidate the mechanism and the transition structure for future inhibitor design studies of TcTS. Additionally, the common inhibitors for sialidases, which catalyze hydrolysis of sialic acids, do not work for TcTS. The reason for this is unclear since both enzyme families share the first step of the mechanism. Trypanosoma rangeli sialidase (TrSA) stands out among sialidases to perform a comparative study with TcTS due to their distinct structural similarity (%70 sequence identity and C-alpha RMSD of 0.59 ?) and yet, different catalytic function. There is experimental evidence about formation of a covalent intermediate in TrSA as well, but only for an activated ligand. Thus, there is a possibility that the mechanism of TrSA is artificially biased towards covalent intermediate formation due to the effect of substituents on the natural ligand. Elucidating the difference in mechanisms of TcTS and TrSA could also pave the way to tailor sialidases into trans-sialidases (and glycosidases into trans-glycosidases) to use for efficient synthesis of molecules that currently require long and low-yield chemical processes. In this study, the mechanisms of both enzymes are investigated using two different QM/MM methods in Chapters 3 and 4. Potential energy surfaces are constructed for each enzyme by performing constrained minimizations. Based on the potential energy surfaces, the difference in the mechanisms of the two enzymes is discussed. Furthermore, 50-ns long molecular dynamics simulations are performed for the two enzymes in free, ligand-bound and inhibitor-bound forms and these simulations are analyzed thoroughly in Chapter 5 to distinguish any structural or dynamical differences between the two enzymes and to shed light on the reason of difference in their inhibitor binding ability.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Ozlem Demir.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Roitberg, Adrian E.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022549:00001


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1 COMPUTATIONAL STUDY OF STRUCTURE AND MECHANISM OF TRYPANOSOMA cruzi TRANS-SIALIDASE By ZLEM DEM R A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 zlem Demir

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3 To my parents, Asiye and Me hmet, and my sister, Glin.

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4 ACKNOWLEDGMENTS I first would like to thank m y supervisor Ad rian Roitberg for all his continuous support, kind guidance, strong encouragem ent and positive feedback thr oughout my graduate studies. His trust, patience and motivation accompanied me all the way, even at the most challenging times. I also wish to thank Nicole Horenstein for valuable discussions and endl ess support. I extend my thanks to all my committee me mbers Nicole Horenstein, Ar thur Edison, Jeffrey Krause, Alexander Angerhofer and former member Nigel Richards for accompanying me in the important stages of my graduate career and all their contributions. Special thanks go to former and present group members Christina Crecca, Seonah Kim, Gustavo Seabra, Georgios Leonis, Hui Xiong, Y ilin Meng, Lena Dolghih, Jason Swails and Dan Sindhikara for all their support. I also want to acknowledge Erik Deumens for his excellent technical help whenever needed and all past and present QTP members and staff for providing a nice and warm environment. This dissertation, like many other nice things in my life, would not be possible without the continuous support of my dear family. I have al ways felt their encouragement and trust on me and I want to thank each of them for all the joy and happiness they brought to my life. It is a place where words are insufficient to express my feelings. It is also hard to acknowledge enough my dear friends in Gainesville who have converted the period I have been here to precious times to remember. I would like to thank Evrim Ata and Avni Argun for their welcoming for all times, c ontinuous support and friendship. Next but not least, I want to thank Asuman and Fatih Grd for being like a second family to me here in Gainesville. It is hard to forget all the good times we shared with dear friends Emine Demir, Esra Byktahtak n, Aysun Altan, Ufuk Koca, Tezcan zrazgat, Emine Dedeo lu, Tuba YavuzKahveci, Yasemin Alptekino lu, Dilber and Dilek Da delen, Fatma Aslan-Tutak, Zeynep opur,

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5 Zeynep rem Atalay, Semra A ral Sevnur Kmrl, Ece nr and Pamela Monterola. Special thanks go to U ur Ba lant Enes al k, Yavuz Ya z, Zafer Demir, Bilge Tutak and Murat Keeli for all their support and friendship. The time I spent here would not be as valuab le without my long-term friends who have been there for me all the time although we were separated by very long distances. Thus, I appreciate and celebrate sincer e friendships of Tuba Glba c Olga Samarskaya, zgr elik, Ebru Yumak, lk Yaylal lk Balc Mine and Abdullah zer.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........9 LIST OF FIGURES.......................................................................................................................10 ABSTRACT...................................................................................................................................15 CHAP TER 1 INTRODUCTION..................................................................................................................17 1.1. Prologue...........................................................................................................................17 1.2. Sialic Acids......................................................................................................................17 1.3. Chagas Disease...............................................................................................................19 1.4. Trypanosoma cruzi ..........................................................................................................21 1.5. Trypanosoma cruzi trans -Sialidase (TcTS) and Trypanosoma rangeli Sialidase (TrSA).................................................................................................................................22 1.5. Structural Information about Trypanosoma cruzi trans-S ialidase and Trypanosoma rangeli Sialidase.................................................................................................................. 26 1.6. Mechanistic Information about Trypanosoma cruzi trans -Sialidase and Trypanosoma rangeli Sialidase...........................................................................................37 2 METHODS.............................................................................................................................44 2.1. Quantum Mechanics........................................................................................................44 2.2. Molecular Mechanics.......................................................................................................47 2.3. Hybrid Methods...............................................................................................................49 2.4. Methods for Exploring th e Potential Energy Surface ...................................................... 51 2.5. Molecular Dynamics Simulation Methods...................................................................... 56 3 QUANTUM MECHANICS/ MOLECULAR MECHANICS STUDY OF THE CATALYTI C MECHANISM OF TRYPANOSOMA cruzi TRANS-SIALIDASE AND TRYPANOSOMA rangeli SIALIDASE USING ONIOM...................................................... 59 3.1. Introduction.............................................................................................................. ........59 3.2. Methods...........................................................................................................................61 3.2.1. Relaxation of the TcTS and TrSA Systems........................................................... 61 3.2.2. Preparation of the TcTS and TrSA Model Systems.............................................. 62 3.2.3. ONIOM Input Setup..............................................................................................63 3.2.4. Potential Energy Surface Scan Setup.................................................................... 64 3.2.4.1. Simulating the trans-sialidas e reaction of TcTS in a SN2 way....................64 3.2.4.2. Simulating the trans-sialidas e reaction of TrSA in a SN2 way....................65 3.2.4.3. Simulating the trans-sialidas e reaction of TcTS in a SN1 way....................65

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7 3.2.4.4. Simulating the sialidase reaction of TcTS................................................... 67 3.3. Results and Discussion.................................................................................................... 67 3.3.1. The SN2-like trans-Sialidase Reaction of TcTS.................................................... 67 3.3.2. The SN2-like trans-Sialidase Reaction of TrSA.................................................... 71 3.3.3. The SN1-like trans-Sialidase Reaction of TcTS.................................................... 73 3.3.4. The Sialidase Reaction of TcTS............................................................................75 3.4. Conclusion.......................................................................................................................79 4 QUANTUM MECHANICS/ MOLECULAR MECHANICS STUDY OF THE CATALYTI C MECHANISM OF TRYPANOSOMA cruzi TRANS-SIALIDASE USING AMBER..................................................................................................................................80 4.1. Introduction.............................................................................................................. ........80 4.2. Methods...........................................................................................................................80 4.2.1. Model Preparation.................................................................................................80 4.2.2. Initial Minimization............................................................................................... 81 4.2.3. QM/MM Potential Energy Surface Preparation.................................................... 81 4.3. Results and Discussion.................................................................................................... 82 4.4. Conclusion.......................................................................................................................85 5 MOLECULAR DYNAMICS STUDY OF TRYPANOS OMA cruzi TRANSSIALIDASE AND TRYPANOSOMA rangeli SIALIDASE................................................... 88 5.1. Introduction.............................................................................................................. ........88 5.2. Methods...........................................................................................................................90 5.2.1. System Setup and Simulation Details.................................................................... 90 5.2.2. Analysis Methods..................................................................................................92 5.3. Analysis...........................................................................................................................95 5.3.1. Stability of the Molecular Dynamics Simulations................................................. 95 5.3.2. Root Mean Square Fluctuation (RM SF) Analysis of MD Si mulations of TcTS and TrSA............................................................................................................96 5.3.2.1. Comparison of RMSF changes due to covalent interm ediate formation in TcTS and TrSA.................................................................................................96 5.3.2.2. Comparison of RMSF between DANA-bound TcTS and TrSA sim ulations............................................................................................................ 98 5.3.2.3. Comparison of RMSF between wild-type TcTS, wild-type TrSA and TrSA5mut simulations............................................................................................ 98 5.3.2.4. Comparison of RMSF between sialyllactose-bound TcTS and sialyllactose-bound TrSA .....................................................................................98 5.3.2.5. Comparison of experimental Bfactors of free and covalent interm ediate forms of TcTS and TrSA.................................................................99 5.3.2.6. Comparison of experimental B-factors between DANA-bound TcTS and TrSA X-ray crystal structures ........................................................................ 99 5.3.2.7. Comparison of experimental B-fact ors between wild-type TcTS, wildtype TrSA and TrSA5mut X-ray crystal structures................................................. 99 5.3.2.8. Comparison of RMSF values from MD simulations and B-factors from X-ray crystal structures ....................................................................................... 103

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8 5.3.3. Comparison of Average Structures from MD Simulations of TcTS in Ligated and Unligated Form s.................................................................................................. 103 5.3.3.1. Comparison of unligated TcTS with TcTS covalent intermediate average structures ...............................................................................................104 5.3.3.2. Comparison of unligated TcTS with DANA-bound TcTS average structures ............................................................................................................. 104 5.3.3.3. Comparison of unligated TcTS with sialyllactose-bound TcTS average structures ............................................................................................................. 107 5.3.4. Comparison of Average Structures from M D Simulations of TrSA in Ligated and Unligated Forms.................................................................................................. 108 5.3.4.1. Comparison of unligated TrSA with TrSA covalent intermediate average structures ...............................................................................................108 5.3.4.2. Comparison of unligated TrSA with DANA-bound TrSA average structures ............................................................................................................. 109 5.3.4.3. Comparison of unligated TrSA with sialyllactose-bound TrSA average structures ............................................................................................................. 109 5.3.5. Comparison of Sialyllactose-Bound TcTS and Sialyllactose-Bound TrSA Average Structures ..................................................................................................... 109 5.3.6. Comparison of DANA-Bound TcTS with DANA-Bound TrSA Average Structures ...................................................................................................................110 5.3.7. Behavior of Key Residues of TcTS and TrSA in MD.........................................110 5.3.7.1. Behavior of Trp312...................................................................................110 5.3.7.2. Behavior of Tyr119...................................................................................114 5.3.7.3. Behavior of Phe58.....................................................................................117 5.3.7.4. Behavior of Asp59....................................................................................117 5.3.7.5. Behavior of Tyr342...................................................................................121 5.3.7.6. Behavior of Glu230...................................................................................121 5.3.7.7. Behavior of Asp96....................................................................................121 5.3.7.8. Behavior of Leu36.....................................................................................122 5.3.8. Ligand-Enzyme Interactions in Ligated TcTS and TrSA MD Simulations........122 5.3.9. Interactions Observed Between the Enzyme Residues in the Active Site ........... 133 5.4. Discussion......................................................................................................................135 5.4.1. Sialyllactose-Bound Form of TrSA..................................................................... 135 5.4.2. Effects of the 5 Point Mutations on TrSA that Prom ote trans-Sialidase Catalysis.....................................................................................................................142 5.4.3. Comparison of DANA-Bound Forms of TcTS and TrSA................................... 144 5.4.4. Comparison of Unligated and DANA-Bound Form s of TcTS............................148 5.4.5. Comparison of the Effect of Covalent Interm ediate Formation in TcTS and TrSA...........................................................................................................................149 CONCLUSIONS AND PERSPE CTIVE..................................................................................... 151 LIST OF REFERENCES.............................................................................................................154 BIOGRAPHICAL SKETCH.......................................................................................................164

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9 LIST OF TABLES Table page 1-1 Sialidase and trans-sialidase activities of wild-type and m utant TcTS and TrSA.............23 1-2 Inhibitor (DANA) binding properties of TcTS, T rSA and several TrSA mutants............ 31 1-3 Trans-Sialidase activity of various TrSA m utants............................................................. 35 1-4 Kinetic constants for the sial idase activity of TrS A mutants............................................. 37 3-1 Results of RC3 scan for TcTS that sh ow stable oxocarbenium ion formation.................. 75 3-2 Results of RC1 scan for TcTS that show conversion of the oxocarbenium ion to the covalent intermediate......................................................................................................... 77 3-3 Results of RC3 scan that show a water molecule attacking the covalent intermediate in TcTS...............................................................................................................................78 4-1 Properties of stationary poi nts on po tential energy surfaces............................................. 83 5-1 Properties of simulated structures...................................................................................... 93 5-2 Hydrogen-bond interactions of the li gands in covalent intermediate, DANA-bound and sialyllactose-bound TcTS. ......................................................................................... 130 5-3 H-bond interactions of the ligands in covalent interm ediate, DANA-bound and sialyllactose-bound TrSA.................................................................................................132 5-4 Hydrogen bonding interactions between th e residues in the active site in MD sim ulations of TcTS species............................................................................................ 134 5-5 Hydrogen bonding interactions between th e residues that lie within 10 of Tyr342.OH atom in MD simulations of free and ligated TrSA species.......................... 135 5-6 Sequence information for different s ialidases and trans-sialidases of Trypanosomal species........................................................................................................................ ......141

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10 LIST OF FIGURES Figure page 1-1 Chemical structures of neuraminic ac id, N-acetyl neuram inic acid and N-glycolyl neuraminic acid................................................................................................................ ..18 1-2 A) Geographical distribution of Chagas disease. B) The insect that transmits Chagas disease: Triatominae C) Romanas sign on the eye, which is a sign of Chagas disease................................................................................................................ ..19 1-3 Different forms of T.cruz i in its life cycle.........................................................................22 1-4 Transfer of sialic acids onto the Trypanosoma cruzi cell su rface...................................... 23 1-5 Sequence alignment of TcTS and TrSA with am ino acid differences highlighted............ 24 1-6 Structures of sialic acid oxocarbenium ion, DANA, oseltam ivir, zanamivir and sialyllactose........................................................................................................................27 1-7 Two different views from superimposed sialyllactose-bound TcTS and unligated TrSA ...................................................................................................................................29 1-8 Active site of TcTS when sialyllactose is bound...............................................................29 1-9 Dual conformations of Tyr119........................................................................................... 30 1-10 The two residues that affect Trp312 in TcTS: Pro283 and Tyr248. .................................. 33 1-11 Sialidase and trans-sialidase activities of chim eric proteins of TcTS and TrSA............... 33 1-12 Different types of bisubstrate enzyme mechanisms........................................................... 38 1-13 Structure of 2,3-difluoro sialic acid used for chem ical trapping.......................................41 1-14 The proposed mechanism for trans-sialidase catalysis r eaction of TcTS. ......................... 42 3-1 The covalent interm ediate for mation step.......................................................................... 65 3-2 Reaction coordinates used to generate potential energy surfaces. ..................................... 66 3-3 Potential energy su rface of TcTS for SN2-like covalent intermediate formation step....... 69 3-4 Changes in geometric prope rties of sialic acid during SN2-like covalent intermediate formation of TcTS..............................................................................................................70 3-5 The potential energy surface of TrSA for SN2-like covalent intermediate formation step.....................................................................................................................................72

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11 3-6 Changes in geometric prope rties of sialic acid during SN2-like covalent intermediate formation of TrSA..............................................................................................................74 3-7 The mechanism proposed for trans-si alidase catalysis reaction of TcTS. ......................... 76 3-8 Potential energy diagram calculated for tr ans-sialidase catalysis reaction of TcTS. ......... 78 3-9 Potential energy diagram calculated fo r sialidase catalysis reaction of TcTS. .................. 79 4-1 Reaction coordinates used to generate potential energy surfaces. ..................................... 83 4-2 Potential energy surface for SN2-like mechanism of TcTS covalent intermediate formation............................................................................................................................84 4-3 Potential energy surface for SN1-like mechanism of TcTS that shows a stable oxocarbenium ion formation..............................................................................................85 4-4 Potential energy surface for SN1-like mechanism of TcTS that shows covalent intermediate formation from the oxocarbenium ion.......................................................... 86 4-5 Potential energy diagram of trans-si alidase reaction of TcTS using QM/MM. .................86 5-1 Corresponding active site residues of TcTS and TrSA that have at least one atom within 10 radius of Tyr342 hydroxyl O atom................................................................90 5-2 The regions of the TcTS and TrSA that are used for MD sim ulation and analysis........... 94 5-3 Description of 1 and 2 dihedral angles of a residue in a protein............................. 94 5-4 Root mean square deviation (RMSD) of MD simulations with respec t to the initial structure of the production runs. A) TcTS species B) TrSA species................................. 97 5-5 C RMSF comparison of free TcTS with TcTS covalent intermediate........................... 100 5-6 C RMSF comparison of free TrSA with TrSA covalent intermediate........................... 100 5-7 C RMSF comparison of free and DANA-bound forms of TcTS....................................101 5-8 C RMSF comparison of free and DANA-bound forms of TrSA.................................... 101 5-9 C RMSF comparison of TrSA, TcTS and TrSA5mut.......................................................102 5-10 C RMSF comparison of sialyllactosebound forms of TcTS and TrSA......................... 102 5-11 Comparison of crystal structure C B-factors of TcTS covalent intermediate with its free form...........................................................................................................................104 5-12 Comparison of crystal structure C B-factors of TrSA covalent intermediate with its free form...........................................................................................................................105

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12 5-13 C B-factor comparison of free and DAN A-bound forms of TcTS X-ray crystal structures..................................................................................................................... .....105 5-14 C B-factor comparison of free and DAN A-bound forms of TrSA X-ray crystal structures..................................................................................................................... .....106 5-15 C B-factor comparison of TcTS, TrSA and TrSA5mut X-ray crystal structures............. 106 5-16 Crystal contacts of TrSA around the loops bearing Trp312, Gln283 and Gly248 .......... 107 5-17 Histograms of Trp312 12 populations in MD simulations for TcTS species............... 112 5-18 Histograms of Trp312 12 populations in MD simulations for TrSA species............... 113 5-19 RMSD of the loop that bears Trp312 in MD sim ulations without fitting to the initial frame................................................................................................................................115 5-20 Correlation of RMSD of the backbone of Trp312s loop with out fitting to the initial confor mation and the 1 dihedral angle of Tyr119 during MD simulations. ................. 116 5-21 Histograms of Tyr119 12 populations in MD simulations..........................................118 5-22 Backbone RMSD for the loop bearing Tyr119/ Ser119 in MD simulations................... 119 5-23 Phe58 1 dihedral angle cha nge in MD simulations .......................................................120 5-24 Asp59 1 change during MD simulations........................................................................ 123 5-25 RMSD of the loop bearing Asp59 in MD sim ulations without fitting............................124 5-26 Histograms of 12 populations of Tyr342 in TcTS MD simulations............................. 125 5-27 Histograms of 12 populations of Tyr342 in TrSA MD simulations.............................126 5-28 dihedral angle of Ala341/Gly341 in MD sim ulations.................................................. 127 5-29 Histograms of 12 populations of Leu36 in TcTS MD simulations.............................. 128 5-30 Histograms of 12 populations of Ile36 in TrSA MD simulations................................ 129 5-31 Hydrogen bonding and steric interact ions in sialyllactose-bound TcTS. ........................131 5-32 Hydrogen bonding and steric intera ctions in sialyllactose-bound TrSA. ...................... 133 5-33 Different conformations of Trp312 seen in TcTS and TrSA. .......................................... 137 5-34 Behavior of Trp312 and its loop backbone in MD sim ulations of A) modeled sialyllactose-bound TrSA and B) sialyllactose-bound TcTS...........................................138

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13 5-35 Close crystal contacts in the unit cell of unligated TrSA. ................................................ 139 5-36 Binding mode of sialyllactos e in A) TcTS and B) TrSA. ................................................141 5-37 A view from superimposed DANA-b ound for ms of TrSA and TcTS average structures of the MD simulations.....................................................................................145 5-38 A view from superimposed DANA-bound Tr SA and TcTS average structures of the sim ulations.................................................................................................................... ...146

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14 LIST OF ABBREVIATIONS DANA 2, 3-dehydro-3-deoxy-N-acetylneuraminic acid PES Potential energy surface RMSD Root mean square deviation RMSF Root mean square fluctuation TcTS Trypanosoma cruzi trans-sialidase TrSA Trypanosoma rangeli sialidase

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15 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy COMPUTATIONAL STUDY OF STRUCTURE AND MECHANISM OF TRYPANOSOMA cruzi TRANS-SIALIDASE By zlem Demir August 2008 Chair: Adrian E. Roitberg Major: Chemistry Trans-sialidase is a vital enzyme for the lifecycle of Trypanosoma cruzi the protozoa responsible for Chagas disease, which is lethal and drastically affects large human populations in Central and South Americawidening its epidem ic area to North Americ a in recent years. T. cruzi trans-sialidase (TcTS) catalyzes transfer of sialic acids from hos t glycoconjugates to the parasites glycoconjugates, whic h facilitates the parasite the means to escape from the host immune system and to invade the host cells. T hus, TcTS stands as a potential and appealing therapeutic target for Chagas disease. Experimental evidence suggests th at a relatively longlived covalent intermediate forms in the mechanism of TcTS. If this scenario is co rrect, sialic acid is s cavenged from the hosts glycoconjugates and stays bound to the enzyme un til the parasites glyc oconjugate enters the active site. However, it is unclear whether the covalent intermedia te formation occurs through an SN1 or SN2 mechanism. It is crucial to elucidate th e mechanism and the transition structure for future inhibitor design studies of TcTS. Additionally, the common inhibi tors for sialidases, which cat alyze hydrolysis of sialic acids, do not work for TcTS. The reason for this is unclear since both enzyme families share the first step of the mechanism. Trypanosoma rangeli sialidase (TrSA) stands out among sialidases

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16 to perform a comparative study with TcTS due to their distinct struct ural similarity (%70 sequence identity and C RMSD of 0.59 ) and yet, differe nt catalytic function. There is experimental evidence about formation of a covale nt intermediate in TrSA as well, but only for an activated ligand. Thus, there is a possibility that the mechanism of TrSA is artificially biased towards covalent intermediate formation due to the effect of substituents on the natural ligand. Elucidating the difference in mechanisms of TcTS and TrSA could also pave the way to tailor sialidases into trans-sialidases (and glycosidases into trans-glycosidases) to use for efficient synthesis of molecules that cu rrently require long a nd low-yield chemical processes. In this study, the mechanisms of both enzy mes are investigated using two different QM/MM methods in Chapters 3 and 4. Potentia l energy surfaces are constructed for each enzyme by performing constrained minimizations Based on the potential energy surfaces, the difference in the mechanisms of the two enzymes is discussed. Furthermore, 50-ns long molecular dynamics simulations are performed for the two enzymes in free, ligand-bound and inhibitor-bound forms and these simulations are analyzed thoroughly in Chapter 5 to disti nguish any structural or dynamical differences between the two enzymes and to shed light on the reason of diffe rence in their inhibitor binding ability.

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17 CHAPTER 1 INTRODUCTION 1.1. Prologue In this chapter, sialic acids, their properties and importance will be introduced first. Then, Chagas disease, one of the many serious diseases that involve sialic ac ids and the key enzymes responsible for sialic acid catabolism, will be described. Brief information about Trypanosoma cruzi which is the causative agent of Chagas diseas e, will follow. Subsequently, the main focus of this study, the trans-sialidase enzyme of Trypanosoma cruzi will be introduced and contrasted to a structurally very similar enzyme, Trypanosoma rangeli sialidase. In the following two sections, current information about structure and mechanism of these two enzymes will be provided in detail. And the last section will in troduce our approach to elucidate the present structural and mechanistic problems of these two enzymes. 1.2. Sialic Acids Sialic acid is the common name for Oa nd N-substituted derivatives of a nine-carbon monosaccharide called neuraminic acid (5 -amino-3, 5-dideoxy-Dglycero-D-galacto-2nonulosonic acid) (Figure 1-1). De rivatives of 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (KDN) which have a hydroxyl group at C5 positio n instead of the amino group of neuraminic acid are also included in this group recently. Sialic acids we re discovered in 1930s and both names, sialic acid and neuraminic acid, reflect the origins of their first isol ation; one was isolated from submaxillary mucin (sialos=saliva in Greek) and the other from brain glycolipids (neuro + amine + acid).1 More than 50 different sialic acids f ound in nature indicate the remarkable structural diversityprovided by substituting th e amino group at C5 position with acetyl or glycolyl groups and/or substituting the hydroxyl groups at C4, C7, C8 and C9 positions with acetate, lactate, sulfate or phosphate esters, or by methyl ethers.

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18 Sialic acids are found in vert ebrate animals, a few invertebrates and microorganisms.2 They lie at the terminal positions of polysaccharide chains, especially in cell membranes, and are used for recognition purposes by the immune system.2,3 Since sialic acid s are strongly acidic with pKa values in the range 2.0-2.6,4 they are negatively ch arged under physiological conditions. Due to their terminal position, sialic acids can both act as masking recognition sites and, conversely, represent specific recognition sites themselves.2 Additionally, due to their negative charge, sialic acids help in exerting attr active/repulsive forces to cells and molecules, and in binding and transport of positively-charged molecules.2 Thus, sialic acids have a significant role in various cell functions, such as host-cell inte ractions like cell adhesion and invasion,5 and resistance to nonspecific complement attack.6 Figure 1-1. Chemical structures of neuraminic acid (Neu, R=H), N-acetyl neuraminic acid (Neu5Ac, R=CH3-CO) and N-glycolyl neuram inic acid (Neu5Gc, R=HOCH2CO). Due to the importance of sialic acids, the sialidase superfamily, that catalyzes hydrolysis of sialo-sugars releasing sialic acids, includes enzy mes that are implicated as virulence factors in pathogenesis of many different diseases.7 For example, the sialidases of avian influenza virus which causes bird flu,8 influenza virus which causes flu,9 paramyxovirus which causes respiratory disease,10-12 Vibrio cholerae which causes cholera, Trypanosoma cruzi which causes

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19 Chagas disease,5 Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense13 which cause sleeping sickness in humans, Trypanosoma brucei brucei14-17 and Trypanosoma congolense13,18 which cause nagana in cattle, all play im portant roles for the mentioned diseases. 1.3. Chagas Disease Chagas dis ease, also known as American trypanosomiasis, is a lethal human disease mostly prevalent in Central and South America and recently shifting its endemic area to North America.19,20 Dating back to ~9,000 years acco rding to the recent evidence,21 Chagas disease is estimated by WHO to have 17 million cases in 21 endemic countries with a death toll of 45,000 people per year due to the cardiac form of the disease (Figure 12.A) and listed as one of the fourteen neglected tropical diseases ( www.who.int/TDR ). Figure 1-2. A) Geographical distri b ution of Chagas disease. B) The insect that transmits Chagas disease: Triatominae C) Romanas sign on the eye, which is a sign of Chagas disease. (Adapted from www.who.int/TDR ) The disease was first described by Dr. Carlos Chagas in 1909 and the causative agent of the dis ease was identified to be Trypanosoma cruzi. 22 Although first observed mostly in rural areas of South America, the disease reached the cities and even to other continents (North America) due to large number s of people emigrating from the endemic countries over the years.19 Unfortunately, there is still no vaccine or e ffective cure after a century and the disease remains as a major medical problem in South America threatening a ve ry large population. The most important precautions for this disease are improving the condi tions of houserendering them unavailable for colonization of the Tria tominaeand performing blood-screening before any blood transfusions. A B C

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20 The current drugs, nifurtimox and benznidazole, are widely used in th e acute phase of the disease but not in the chronic phase.23 In addition to having severe side effects including anorexia, loss of weight, psychic alterations, excitability, sleepine ss, nausea, skin manifestations like hypersensitivity, depression of bone marrow, these drugs harm the adrenal glands, oesophagus, colon and reproduc tory and mammary tissues.23 They also show neurotoxicity, mutagenetic and carcinogenic effects.23 All these side effects gene rally cause discontinuation of their usage by the patients. Thus, effective dr ugs for Chagas disease are urgently needed. Chagas disease can be transmitted to humans by several known ways: (1) Being bitten by bloodfeeding Triatominae vectors (Figure 1-2.B) (2) Transfusion of infected blood24 (3) Transplant of an infected organ25,26 (4) Congenitally, from mother to the baby27 (5) Breastfeeding28 (6) Using food or drinks co ntaminated with crushed Triatominae vectors29 The first one is the most common way of transm ission responsible for 80-90% of all cases. Six to ten days after infection, the clinical signs of immediate acute stage of the disease start to appear and these can continue up to 2 months.30 The acute stage typically causes oedema at the infected locationcalled Romanas sign if at the eyelid (Figure 1-2.C)due to rapid reproduction of causative agent of the disease and th e lack of the immune response. It is also possible that no noticeable sign of this stage is seen. The acute stage is over once a balance between the host and the parasite is reached due to the immune system reducing the number of parasites in the circulatory system. The subs equent chronic stage c ontinues life-long and can seriously damage the heart, oesoph agus, colon and the nervous system.30 Megaoesaphagus, megacolon, heart failure and sudde n death are often seen among the patients. The infected people and vertebrates serve as a potential reservoir since Triatominae biting them can transmit the parasites to their future victims.

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21 1.4. Trypanosoma cruzi The causativ e agent of Chagas disease, Trypanosoma cruzi is a flagellatae protozoan. The lifecycle of this parasite involve s vertebrate hosts and bloodfeedi ng insects of Triatominae (a.k.a. kissing bugs), which is a subfamily of Reduviidae.31 The most important of the species that transmit Chagas disease are Triatoma infestans, Rhodnius pr olixus, Triatoma dimidiata, Triatoma brasiliensis and Panstrongylus megistus Most species of Triatominae live on wild nesting vertebrates and only a few species are associated with humans and domestic animals. These insects feed at night when the hosts are asleep and they leave their feces that include T. cruzi parasites around the bite wound during or right after getting the bloodmeal from the host. The parasites are carried into the body through the wound when the host scratches the wound due to intensive itching. T. cruzi is in epimastigote form when it first ente rs the insect vector by way of a bloodmeal from an infected host (Fi gure 1-3). In this stage, T. cruzi does not express trans-sialidase and has no sialic acids on its cell surface. After several changes in the gut of the insect, the parasite moves to the rectal gland of the insect where it multiplies and produces infective metacyclic trypomastigote forms of T. cruzi ready to leave the insect with defecation. The metacyclic forms express trans-sialidase and once transmitted to the human (or any vertebrate host) by the insect vector by way of a bloodmeal, th ey acquire sialic acids from the host sialoglycoconjugates and are able to invade cells in the host body.5 Soon after invasion by the parasite, the membrane of the invaded host cell is ruptured and the parasi tes are exposed to the cytoplasm where they transform into amastigote form that can multiply very quickly. Amastigotes grow and transform into trypomastigote form. Trans-sialidase expression is found to be stage-specific and turned on only during metacyclic stages.32,33

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22 As with many other parasites, the presence, quantity and localization of sialylated glycoconjugates on the cell surface is very impor tant for the survival and infectivity of T. cruzi. Since it is unable to synthesize sialic acids,34,35 Trypanosoma cruzi depends on its trans-sialidase enzyme to acquire sialic acids from host sialoglycoconjugates (Figur e 1-4). Sialic acids transferred to its surface glycoc onjugatescalled mucinsprovide T.cruzi the ability to evade the immune system of the host and to adhere to and invade the host cells.6,36 The parasite invasion is found to be reduced significantly wh en sialylated epitopes are neutralized with antibodies,5 when trans-sialidase is neutra lized by various other protocols37-39 or when sialic acids are present in neither the hos t cells nor the external medium.40 The direct involvement of trans-sialidase in animal pathogenesis41 and thymocyte apoptosis42 is also shown. Figure 1-3. Different forms of T.cruzi in its life cycle (Adapted from Figure 2 in Tyler et al. 33) 1.5. Trypanosoma cruzi trans-Sialidase (TcTS) and Trypanosoma rangeli Sia lidase (TrSA) Sialidase superfamily catalyzes hydrolysis of sialo-sugars releasing sialic acids and includes enzymes that act as virulence factor s in pathogenesis of ma ny different diseases.7 Trypanosoma cruzi trans-sialidase (TcTS), a member of th is family, is much more efficient in catalyzing transfer of sialic ac ids in the presence of suitable sugar acceptors (glycoconjugates with terminal -galactose) than hydrolysis (Table 1-1).32,43-46 T. cruzi the parasite responsible for Chagas disease, is unable to synthesize sialic acids de novo34 but expresses TcTS to evade

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23 immune response of the host and to invade cells.5,32,42,47 The role of TcTS in survival and infectivity of T.cruzi besides the lack of this enzyme in mammals makes it a potential and appealing therapeutic target. Figure 1-4. Transfer of sialic acids onto the Trypanosoma cruzi cell surface. Table 1-1. Sialidase and trans-sialidase activi ties of wild-type and mutant TcTS and TrSA.48 Sialidase activity Trans-sialidase activity Substrate: -2,3-sialyllactose TcTS wild type 306.0 4.4 1412.98 21.95 TcTS Trp312Ala mutant 185.1 7.1 0 TrSA wild type 4298.7 34.4 0 TrSA Trp312Ala mutant 3567.8 56.9 0 TrSA Gln283Pro mutant 11655 95 0 Substrate: -2,6-sialyllactose TcTS wild type 0 0 TcTS Trp312Ala mutant 109.9 8.3 0 TrSA wild type 0.7 0.2 0 TrSA Trp312Ala mutant 72.8 11.1 0 Activities are expressed as nmol sialic acid (free sialic acid for sialidase activity, sialic acid transferred to lactose for trans-sialidase activity) min-1mg-1. TcTS specifically cata lyzes transfer of -2, 3-linked N-acetylneuraminic acids (which will be referred to as sialic acid from now on th roughout this study) to ac ceptors with terminal galactosyl residues with re tention of configuration.49 Thus, TcTS differs from sialyltransferases which cause inversion of configuration and require sugar nucleotides like cytidine

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24 Figure 1-5. Sequence alignment of TcTS and Tr SA with amino acid differences highlighted. TcTS 1 ---LAPGSSRVELFKR QSS K VPFEK-DG KVTERVVHSFR L P ALVNVDGV TrSA 1 AASLAPGSSRVELFKR KNS T VPFEESNG TIRERVVHSFR I P TIVNVDGV TcTS 46 MVAIADARYETS N DNSL I D T VAKYSVDDG E TWE TQIAIKNSRASSVSRV TrSA 50 MVAIADARYETS F DNSF I E T AVKYSVDDG A TWN TQIAIKNSRASSVSRV TcTS 95 V D P TVIVKGNKLY V LVGSY N SSR SYWTS H G D ARDWDILLA VGEVTKS T A TrSA 99 M D A TVIVKGNKLY I LVGSF NKTRNSWTQ H R D GSDWEPLLV VGEVTKS A A TcTS 144 G GKI TAS I K WGS PVSLKEFFPAEMEG MHT NQF L GGA G V AIVASNGNLVY TrSA 149 N GKT TAT I S WGK PVSLKPLFPAEFDG ILT KEF V GGV G A AIVASNGNLVY TcTS 192 PVQ VTNKKKQ VFS KIF YSEDE G K TWKFGKGRSA FGCSEP VALEWEGKLI TrSA 196 PVQ IADMGGR VFT KIM YSEDD G N TWKFAEGRSK FGCSEP AVLEWEGKLI TcTS 242 IN T RVDYRRRLVYE TWDMGN TWL EAV GTLSR VWGPSPK SNQPGSQSSFT TrSA 246 IN N RVDGNRRLVYE SSDMGK TWV EAL GTLSH VWTNSPT SNQQDCQSSFV TcTS 291 AVTIEG M RVMLFTHPLN F KGRWL RDRLN LWL TDNQRI YNVGQV SIGDEN TrSA 295 AVTIEG K RVMLFTHPLN L KGRWM RDRLH LWM TDNQRI FDVGQI SIGDEN TcTS 340 S A YSSVLYKDDKLY C LHEINS N E VYSLVF A RLV GELRIIKSVLQSWKNW TrSA 344 S G YSSVLYKDDKLY S LHEINT N D VYSLVF V RLI GELQLMKSVVRTWKEE TcTS 389 D S HLS SICTPADPAASSS ERGCGP AVT T V GLVGFLSHSA TKTEWEDA YR TrSA 393 D N HLA SICTPVVPATPPS KGGCGA AVP T A GLVGFLSHSA NGSVWEDV YR TcTS 438 CV N A STANAERVPNGLKF A GVGGGA L WPVSQQGQNQRYH FANHAFTLVA TrSA 442 CV D A NVANAERVPNGLKF N GVGGGA V WPVARQGQTRRYQ FANYRFTLVA TcTS 487 S VTIH E V PKGA SPLLGA S L DSSG GKKLLGLSYDK RHQWQ P I YGSTP VTP TrSA 491 T VTID E L PKGT SPLLGA G L EGPG DAKLLGLSYDK NRQWR P L YGAAP ASP TcTS 537 TGSWE MGK R YHVVLTMA NKIGSVYI DGE PLE GSGQ TVVPDERTPDISHF TrSA 541 TGSWE LHK K YHVVLTMA DRQGSVYV DGQ PLA GSGN TVVRGATLPDISHF TcTS 585 Y V GGYKRSG M PTDSRVTV N NVL LYNRQ LNAEEIRTLFLSQD L IGTE TrSA 589 Y I GGPRSKG A PTDSRVTV T NVV LYNRR LNSSEIRTLFLSQD M IGTD

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25 monophosphate (CMP)-N-acetylneuramin ate as sialic acid donors.5,50-54 Recently, it was shown that TcTS can also ca talyze the transfer of -2, 3-linked N-glycolylneuraminic acid to acceptors with terminal -galactosyl residues, even more efficien tly than the transfer of sialic acids.55 The enzyme is not sensitive to the monosaccharide linked to -galactose; open chain derivatives of lactose, like lactitol and lactobionic acid, ar e also found to be good acceptors of sialic acid.56-58 TcTS is a glycosylphosphatidilinositol(GPI)-anc hored surface enzyme that consists of a 70 kDa globular core that accomm odates the catalytic site and a variable number of highly immunogenic repeats termed SA PAshed acute phase antigen.43 The enzyme has an optimum activity around pH 7.9.46 About 140 genes in the T.cruzi genome encodes for TcTS protein family of which most members lack catalytic activity due to a key mutation (Tyr342His).59-61 TcTS belongs to the family of exo-sialidases (EC 3.2.1.18)62,63 and is listed as a member of glycoside hydrolase family GH 33 according to the Henrissat classification (www.cazy.org ).63 A closely related parasite, Trypanosoma rangeli, expresses a surface sialidase which lacks SAPA tail but has unusual similarity to 631-residue-long globular core of TcTS. T.rangeli coexists with T.cruzi sharing the same vector host specie s and epidemic area and thus, makes it harder to diagnose Chagas dis ease and to distinguish from T.rangeli infections.64 Trypanosoma rangeli sialidase (TrSA) has an optimum activity around pH 5.565 and is also encoded by a multi-gene family some members of which encode for catalytically inactive enzymes.66-68 There are 5 glycosylation sites in th e natural TrSA enzyme at asparagine residues 14, 23, 114, 428 and 614 which are proven to have no role in catalysis since recombinant TrSA lacking these sugar residues also shows the same enzymatic activity.69 The sequence identity is 70% between Tr SA and TcTS with only four amino acid insertions in TrSA close to the N-terminus (Figure 1-5). One can ev aluate the extent of similarity

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26 comparing with the fact that any two proteins having more than 30% sequence identity are known to adopt essentially the same structure.70 When TcTS and TrSA are superimposed using corresponding C atoms, the root mean square deviati on (RMSD) is only 0.59 The active sites of the two enzymes are also very similar as w ill be explained in deta il in the next section. Despite this structural similarity, TrSA is found to lack any trans-sialidase activity48,66,71 which makes these two enzymes an excellent case study to elucidate the reasons for the two different catalytic activities (Table 1-1). Another important difference of TcTS from TrSA is in their inhibitor binding properties. 2,3-dehydro3-deoxy-N-acetylneuraminic acid (DANA) ( 3, Figure 1-6), which is a structural analog of sialic acid oxocarbenium ion ( 2, Figure 1-6) and an efficient inhibitor for TrSA and sialidases in general, does not inhibit TcTS44,72(Table 1-2). Other sialidase inhibitors like oseltamivir ( 4 Figure 1-6) and zanamivir ( 5, Figure 1-6) do not inhibit TcTS, either. With such similar active sites, the reasons of this inhibition difference await deciphering and can guide inhib itor design studies for TcTS. Unraveling the minute but vital differences be tween TcTS and TrSA could also pave the way to tailor sialidases into trans-sialidases (a nd glycosidases into trans-glycosidases) to use for efficient synthesis of oligosaccharides with terminal sialic acids.72-74 Using modified glycosidases instead of long and low-yield chemical processes to synthesize desired polysaccharides will be a great improvement if th e large number of various glycosidases can be tailored into efficient trans-glycosidas es that are not naturally available. 1.5. Structural Information about Trypanosoma cruzi trans-Sialidase and Trypanosoma rangeli Sialidase Due to its importance for Chagas d isease, Tc TS is one of the most extensively studied enzymes among sialidases. The X-ray crystal structures of free and DANA-bound forms of TcTS were elucidated in 200275 just after those of TrSA were obtained.69,76 The structures of Michaelis

PAGE 27

27 Figure 1-6. Structures of si alic acid oxocarbenium ion ( 2), DANA ( 3), oseltamivir (4 ), zanamivir ( 5) and sialyllactose ( 6). The monosaccharide units of sialyllactose are also shown. 2 3 4 5 6

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28 complex of TcTSAsp59Ala mutant with sialyllactose and the co valent intermediate forms of both TcTS and TrSA using activated sialic acid substrates were also obtained in subsequent crystallographic studies.77,78 Both enzymes consist of two gl obular domains that are attached by a long -helical segment (Figure 1-7). Equivalent pos itions in the two enzymes differ by 4 amino acids because of four residue insertions pres ent in TrSA in the fi rst 25 residues of the Nterminus. The residue numbering in TcTS will be referred for equivalent positions of both enzymes to keep consistency throughout this work unless otherwise stated. The N-terminal domain (residues 1-371) has a six-bladed -propeller topology similar to bacterial and plant sialidases and is where the active site resides. An -helical region (residues 372-395) connects the N-terminal domain to the C-termin al domain (residues 396-640) which has a -barrel topology similar to plan t lectins and a short -helical region at the end. The narrow catalytic cleft seen in the crystal structures argues against the formerly suggested sequential mechanism46,79 which requires concomitant binding of the donor and the acceptor substrates to the enzyme. The catalytic cleft of TcTS consists of tw o sites; the sialic acid binding site which accommodates the sialic acid part of the donor liga nd and is buried deep in the enzyme and the aglycon binding site which accommodates the lact ose part of the donor ligand and subsequently the acceptor ligand (lactose, 6 in Figure 1-6, or any sugar with a terminal -galactose) (Figure 18). The aromatic side chains of Trp312 and Tyr119 lie parallel to each other and form the two lateral walls of the aglycon bi nding site at the periphery of TcTS. Although both of these two residues lie on flexible loops, both loops adopt the same pose in all X-ray crystal structures of TcTS and TrSA. Another interesting property of Tyr119 depicted in Figure 1-9 is that two different conformations of Tyr119 are observed unless the aglycon bi nding site is filled with a ligand. In one conformation (the down confor mation), Tyr119 side chain swings into the

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29 Figure 1-7. Two different view s from superimposed sialyl lactose-bound TcTS (red) and unligated TrSA (blue). Sialyllactose is shown in orange. Figure 1-8. Active site of TcTS when sial yllactose (in CPK form) is bound. A) Trp312 and Tyr119 that lie at the periphery of the activ e site and surround the lactose part of the ligand. The arginine triad that interacts with the carboxylat e group of the sialic acid part of the ligand is also shown. B) The catalytic nucleophile, Ty r342/Glu230 pair, lie at the bottom of the catalytic cleft while Asp59 interacts wi th the glycosidic O atom that connects sialic acid and lact ose parts of the sialyllactose. A B Tyr342 Trp312 Tyr119 Glu230 Asp59 Arg245 Arg314 Arg35 Glycosidic O

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30 catalytic cleft interacting with Asp96, Gln195 and Glu230 and in the presence of sialic acid also with the glycerol side chain of sialic acid, while in the other conformation (the up conformation) Tyr119 side chain completely leav es the catalytic cleft extending into the bulk solvent. Emphasizing the role of hydrophobic in teractions with Tyr119 and Trp312, the aglycon binding site does not have any di rect hydrogen bonding interactions with the lactose moiety of the ligand, but water-mediated interactions in stead, except the interaction of Asp59 carboxylic acid with the glycosidic oxygen77(Figure 1-8). Figure 1-9. Dual conformations of Tyr119. The sialic acid binding site of TcTS catalytic cleft shares se veral features with microbial sialidases7,80(Figure 1-8). The arginine triadA rg35, Arg245 and Arg314 that forms strong salt bridges with the carboxylate group of sialic acid, glutamate (Glu357) that stabilizes Arg35, Asp59 that acts as an acid/base catalyst and a tyrosine and a glutamic acid residue (Tyr342 and Glu230) that lie at the bottom of the catalytic cleft and act as a nucleophile couple in microbial sialidases are all conserved in TcTS. Trp312 Tyr119 Tyr342

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31 The active site of TrSA is very similar and has the same residues mentioned so far in TcTS active sitein very similar positionsexcept having a serine resi due instead of Tyr119 residue. Due to this difference, TrSA lacks the second late ral wall seen in TcTS that embraces the lactose part of the ligand and thus, the active site of TrSA is more so lvent-exposed. However, a simple Ser119Tyr mutation of TrSA does not provide it with any trans-sia lidase activity, so the story is clearly more complex.Taking advantage of the very few amino acid differences close to the active sites of TcTS and TrSA, a number of mutagenesis studies ha ve been performed to unravel the critical amino acids necessary for trans-sialidase activity.48,69,81 Table 1-2. Inhibitor (DANA) bi nding properties of TcTS, TrSA and several TrSA mutants72 Ki (mM) TcTS wild type 12.29 TrSA wild type 0.0015 TrSA5mut 1.54 TrSA5mut Ile37Leu 1.01 TrSA5mut Gly342Ala 0.54 Mutagenesis studies showed the importance of Tyr119 and Trp312 for trans-sialidase catalysis in TcTS. Tyr119 in TcTS is a serine residue in TrSA and thus, could very well be a reason for the trans-sialidase ability of TcTS. 48,69,81 However, TrSA Ser119Tyr mutant failed to catalyze sialic acid transfer while the inverse mutation (Tyr119Ser) caused TcTS to lose almost all its trans-sialidase ability.48 These results show that presence of Tyr119 residue is necessary but insufficient by itself for trans-sialidase ab ility. It is also shown that Trp312Ala mutation completely abolished trans-sialidase activity of TcTS while the same mutation only decreased sialidase activities of TcTS and TrSA indicating the impor tance of Trp312 for trans-sialidase activity48 (Table 1-1). Trp312 is also found to be the reason of high substrate specificity of TcTS and TrSA for sialyl-2,3-linked-oligosaccharides since Trp312Ala mutants of both TcTS and TrSA can additionally hydrolyze sialyl-2,6-linked-olgosaccharides which can not be

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32 hydrolyzed by the wild type enzymes at all48 (Table 1-1). Sialidases of Vibrio cholerae Micromonospora viridifaciens and the influenza virus which have dual -(2, 3) -(2, 6) substrate specificity all have a shorter loop in the corr esponding regionconstituting a completely solvent-exposed cat alytic cleftand no tryptophan residue like Trp312 confirming the role of Trp312 in substrate specificity.69 Even the two residues Pro283 and Tyr248 that lie side by side on two loops neighboring the loop of Trp312 in TcTS prove to be important for trans-sialidase activity since both residues sterically help Trp312 side chain to adopt a suitable conformation for binding (Figure 1-10). These two residues are among the critical amino acid differences between TcTS and TrSA; Pro283 and Tyr248 in TcTS correspond to a glutamine and a glycine residue, respectively, in TrSA. However, TrSAGln283Pro and even TrSAGln283Pro/Gly248Tyr mutants did not show any trans-si alidase activity, but they both showed significantly increased sialidase activities.48 Curiously, both trans-sialidase and sialidase activities are abolished in TcTSPro283Gln mutants.48 Even the double mutant TcTSTyr248Gly/Pro283Gln and the triple mutant TcTSTyr119Ser/Tyr248Gly/Pro283Gln lack both trans-sialidase and sialidase activities. All these mutagenesis results point to the necessity of the precise location of Trp312 si de chain to lock the lactose part of the ligand (especially the acceptor ligand which binds the enzyme only through this interaction) into a pr oper conformation for trans-sial idase catalysis in TcTS. A later study further confirmed the importa nce of Pro283 residue for trans-sialidase activity. Since most of the residues related to catalysis reside among the first 200 residues, chimeric enzymes were constructed exchanging the 200-residue long segment of N terminus of TcTS and TrSA and their sialidase and trans-sial idase activities were measured (Figure 1-11).

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33 Figure 1-10. The two residues that af fect Trp312 in TcTS: Pro283 and Tyr248. Catalytic domain Lectin-like domain 200 200 200 200 Pro283 Gln283 Pro283 Pro283 Pro283 Gln283 Gln283 Activity SA TS + + + + + + + --TcTSw.t. TrSAw.t. TrSA Gln283Pro TcTSw.t./ TrSAw.t. TcTSw.t./ TrSAGln283Pro TrSAw.t./ TcTSw.t. TrSAw.t./ TcTSPro283Gln Catalytic domain Lectin-like domain 200 200 200 200 Pro283 Gln283 Pro283 Pro283 Pro283 Gln283 Gln283 Catalytic domain Lectin-like domain 200 200 200 200 Pro283 Gln283 Pro283 Pro283 Pro283 Gln283 Gln283 Activity SA TS + + + + + + + --TcTSw.t. TrSAw.t. TrSA Gln283Pro TcTSw.t./ TrSAw.t. TcTSw.t./ TrSAGln283Pro TrSAw.t./ TcTSw.t. TrSAw.t./ TcTSPro283Gln Figure 1-11. Sialidase and trans-sialidase activitie s of chimeric proteins of TcTS and TrSA. The blue and white bars represent TcTS and TrSA-derived sequences, respectively. The amino acid identity at pos ition 283 is also indicated. Tyr248 Pro283 Trp312 Tyr119

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34 The chimera with the N-terminus of TcTS and th e C-terminus of TrSA was found to be totally inactive, while the other chimera with the N-terminus of TrSA a nd the C-terminus of TcTS had a strict sialidase activity. An additional Gln283Pro mutation was found to confer trans-sialidase activity to the second chimera with strict sialidase activ ity (Figure 1-11). Unable to confer trans-sialid ase activity to TrSA with singl e, double or triple mutations, other possible mutations in TrSA active site to achieve this goal were investigated. For this purpose, a sequence alignment of TcTS, TrSA and Trypanosoma brucei trans-sialidase (TbTS) which has only about 30% sequence similarity to TcTS and TrSA was al so referred as a guide.72 The three essential mutations for trans-sialidase activity identified in previous studies (Ser119Tyr, Gly248Tyr, Gln283Pro) were supplemented with two mutations in TrSA: Met95Val and Ala97Pro. These two mutations were chosen for several reasons. First, they are both neighbors of a conserved residue in the act ive site (Asp96). Second, Val95 and Pro97 are conserved both in TcTS and TbTS while thes e two residues are substituted with Met95 and Ala97 in TrSA. And third, different interacti ons of Asp96 with the inhibitor DANA are observed in crystal structures of DANA-bound forms of TcTS and TrSA probably related to the identity of neighboring residues. The Tr SA quintet mutant (TrSA5mut) prepared using this strategywith Ser119Tyr, Gly248Tyr, Gln283Pro, Met95Val and Ala97Pro muta tionsachieved to confer trans-sialidase activity in TrSA scaffold for the first time alth ough it is only 1% of the transsialidase activity of wild type TcTS72 (Table 1-3). In order to incr ease the trans-sialidase activity of TrSA5mut additional mutations were introduced as shown in Table 1-3. Among these Gly341Ala and Ile36Leu mutations prove to be effective significantly obtaining 10% of the trans-sialidase activity of TcTS. Both of these mutations were chosen due to their possible effect on the nucleophile, Tyr342. Additionally, Gly341 (Ala341 in TcTS) probably affects the

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35 flexibility of Tyr342 while Ile36 (Leu in TcTS) is in contact with the side chain of Tyr342 and has alternate conformations. The sialidase activi ties of all the mentioned mutants were also determined in the same study.72 As shown in Table 1-4, single Gln283Pro mutation promoted the sialidase activity of TrSA si gnificantly, which further confir med the importance of Pro283. TrSA5mut and its further mutated forms which have ac quired trans-sialidase activity all showed low sialidase activities (Table 1-3, Table 1-4). Al so, the insensitivity of the enzymes to inhibition by DANA was observed to increase as they acquire trans-sialidase activity for TrSA5mut and its further mutated forms, however, it still did not reach the level of TcTS (Table 1-2). DANA-bound forms of TcTS and TrSA are found to differ in the enzyme-ligand interactions seen in X-ray crystal structures. The hydroxyl groups of the glycerol moiety of DANA form several direct hydrogen bonds to Tc TS residues while only water-mediated bonds between this region of DANA and TrSA residues are observed.76 The O4 hydroxyl group of DANA interacts with Asp59 in TcTS while it interacts with Asp96 in TrSA. Table 1-3. Trans-sialidase activity of various TrSA mutants72 Protein TS activity (pmol min1 g protein-1)a App Km (mM)b App Vmax (pmol min1 g protein-1)b TrSA wt/TrSA Ser119Tyr/TrSA Gln283Pro/TrSA Met95Val, Ala97Pro/TrSA Gly248Tyr, Gln283Pro/TrSA Ser119Tyr, Gly248Tyr, Gln283Pro Undetectable TrSA5mut 3.84 0.18 (0.9) TrSA5mut Ile36Leu 47.20 5.50 (11.3) 9.44 2.00 14.70 1.11 TrSA5mut Gly341Ala 45.28 3.30 (10.9) 6.90 2.57 6.39 1.30 TrSA5mut Val179Ala 10.15 0.21 (2.4) TrSA5mut Phe113Tyr 11.11 0.57 (2.7) TrSA5mut Thr38Ala 26.62 0.72 (6.4) TrSA5mut Asp284Gly 12.13 0.78 (2.9) TcTS wt 416.71 47.43 (100) 0.24 0.03 3.83 0.42 a The activity is measured using sialyllactose and lactose as donor and accep tor, respectively. The percentage of trans-sialidase activity compared to w ild type TcTS is given in parenthesis. b Apparent kinetic constants for lactose are determined keeping sialy llactose at 2 mM concentration and varying the lactose concentration.

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36 The process of trying to obtain X-ray crystal structure of TcTS gave additional structural clues. They found that the crystals of TcTS b ecome instable and crack readily when DANA or sialic acid is bound to it.75 In addition, TcTS tends to prec ipitate when it is incubated with DANA or sialic acid.75 Both of these observations suggest that conformational changes occur upon ligand binding, which ultimately disrupt the molecular packi ng of crystals. In the same study, they also found that soaking unligated TcTS crystals with lactose failed to produce any lactose-bound TcTS crystals.75 Since lactose is a natural accepto r ligand for the transfer reaction of TcTS, it definitely can bind TcTS. To elucidate this issue, surface plasmon resonance experiments were performed on the immobilized TcTSin an enzymatically inactive form due to Asp59Asn mutationusing sialy llactose and lactose as mobile specific ligands separately.75 No lactose binding to TcTS could be detected even at very hi gh concentrations like 100 mM while sialyllactose binding to TcTS was detected even in the concentration range of 0.1-2 mM. However, if the same experiment was performed after TcTS was preequilibrated in a buffer that contains sialic acid or sialyllactose, lactose binding to TcTS was clearly detected at 10 mM concentration. These results clearly demonstrate that the affinity for the acceptor substrate is modulated by donor substrate binding, probably due to a structural change. Another result obtained from the X-ray crysta llography studies is that TcTS can produce DANA. All TcTS crystals soaked with sialic acid ended up having DANA in their active sites.75 This was also observed previously in a different study by Todeschini et al .49 The structural and dynamical properties of Tc TS and TrSA will be thoroughly inquired in Chapter 5 by the analysis of molecular dynamics simulations of the two enzymes.

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37 Table 1-4. Kinetic constants for the sialidase activity of TrSA mutants72 Protein Km (mM)a Vm (nmol min-1 g protein-1)a kcat (s-1)b 10-5*kcat/Km (M-1s-1)b TrSA wt 0.273 0.010 273.11 .82 151.42 5.55 TrSA Ser119Tyr 0.068 0.002 45.48 .17 27.21 3.98 TrSA Gln283Pro 0.082 0.02 1175.14 27.60 703.03 86.08 TrSA Met95Val,Ala97Pro 1.079 .048 285.98 .40 171.09 1.59 TrSA Gly248Tyr,Gln283Pro 0.070 .001 225.76 .57 135.06 19.2 TrSA Ser119Tyr, Gly248Tyr,Gln283Pro 0.122 .008 159.32 .33 95.31 7.83 TrSA5mut 0.039 0.001 71.54 1.32 42.80 10.88 TrSA5mut Ile36Leu 0.200 .009 106.04 .43 63.44 3.17 TrSA5mut Gly341Ala 0.107 .003 38.29 .87 22.91 2.13 TrSA5mut Val179Ala 0.172 .029 49.48 .18 29.60 1.73 TrSA5mut Phe113Tyr 0.038 0.001 74.91 1.53 44.82 11.79 TrSA5mut Thr38Ala 0.029 0.001 56.03 1.17 33.52 11.65 TrSA5mut Asp284Gly 0.025 0.001 52.85 0.92 31.62 12.70 TcTS wt 0.291 0.022 0.30 0.022 0.18 0.0062 Molecular mass of TcTS and TrSA are 71.2 kDa and 76.1 kDa, respectively. Sialidase activities are determined using 2-(4-methylumbelliferyl)-D-N-acetylneuraminic acid as substrate and fluorescence measurements of released 4-methylumbelliferone. a Km and Vm values are estimated using a Lineveawer-Burk plot. b kcat and kcat/Km values are obtained using the Km and Vm values in the previous columns. 1.6. Mechanistic Information about Trypanosoma cruzi trans-Sialidase and Trypanosoma rangeli Sialidase The m echanism of Trypanosoma cruzi trans-sialidase (TcTS) is extensively studied due to its role in Chagas disease. Both sialidase and trans-sialidase reactions of TcTS have been explored. Kinetic isotope effect studies, initial velocity studies, chemical trapping and chemical rescue experiments are examples of experimental methods utilized. There are two possible mechanisms for bi substrate enzymatic reactions; sequential mechanism and ping-pong mechanism. The se quential mechanism requires binding of both substrates to the enzyme before any chemi cal reaction happens, while the ping-pong mechanism requires chemical reaction to occur after the bind ing of the first substr ate that results in a modified form of the enzyme, which subsequen tly binds the second substrate and completes the reaction (Figure 1-12). The sequential mechanis m has two kinds; ordered sequential mechanism and random sequential mechanism. Ordered sequen tial mechanism requires binding of one of the

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38 substrates particularly before the other one, wh ile in a random sequential mechanism the order of binding of the substrates does not matter. Figure 1-12. Different types of bisubstrate enzyme mechanisms. A and B represent the substrates while P and Q represent the products. E is modified fo rm of enzyme E due to covalent bonding to a ligand. To get detailed information about TcTS mechanism, primary and -2H kinetic isotope effects (KIE) were determined both in TcTS and contrasted with the ones in acidic solution.82 Primary 14C (or 13C) KIEs give a measure of the associa tive/dissociative charac ter of the reaction while -2H KIEs inform us about the charge developm ent at the anomeric carbon (Figure 1-1) in the transition state. The data fo r the acid-catalyzed solvolysis reaction of the substrate were indicative of a transition state w ith a dissociative char acter (with very lit tle, if any, nucleophilic participation) and a significant charge devel opment at the anomeric carbon. However, large primary 14C KIEs and small -2H KIEs measured show that the transition state in the transsialidase reaction of TcTS has an associativ e character (i.e. has significant nucleophilic participation) with little charge developmen t at the anomeric carbon. The information obtained about the transition state for tr ans-sialidase reaction of TcTS pointed towards a subsequent formation of a covalent intermediate. Additionally, very similar -2H KIEs obtained in the same A P B Q A B P Ping-pong mechanism Sequential mechanism Q E E E E EA EP EB EQ E EAB EPQ

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39 study at different concentrations of the acceptor s ubstrate ruled out the possibility of sequential ordered mechanism for TcTS that wa s suggested based on previous data.46,79 Initial velocity studies of the trans-sialidas e reaction of TcTS were also performed by the same researchers to complement the KIE studies.83 The initial velocity results can distinguish sequential and ping-pong mechan isms; the double reciprocal pl ot (1/V vs. 1/[Substrate1] while [Substrate2] is held constant) consists of intersecting lines fo r a sequential mechanismboth random and ordered ones, while parallel lines are typical for a ping-pong mechanism. The pattern for a ping-pong mechanism can change if a ny branch reactions exis t, like the hydrolysis reaction in TcTS, but one can still distinguish a ping-pong mechanism from a random sequential mechanism by measuring initial ve locities of both the first an d second product formation. The double reciprocal plot obtained monitoring the first product formation of TcTS reaction showed parallel lines. However, the double reciprocal plot obtained by monitoring the second product formation resulted in intersecting lines when performed in low substrate concentrationsat which the hydrolysis reaction becomes significan t. These data indicated the mistake of the previous steady-st ate kinetic studies46,79 that were interpreted as implying a sequential mechanism for TcTS without taking into account the hydrolytic branch re action of TcTS. Thus, a branched ping-pong mechanism was suggested by this study for TcTS. To further the analysis, a chemical trapping experiment with radioisotope -labeled sialyllactose as the substrate was performed, which successfully captu red a labeled intermediate w ith sialic acid covalently bound to the enzyme.52,83 This study was the first to obtain evidence for a ping-pong mechanism including covalent intermediate formation, much before any structural data from X-ray crystallography became available. Although it was not possible to identify the residue of TcTS acting as the nucleophile, they corr ectly predicted it to be Tyr342.

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40 The X-ray crystal structures of TcTS were obtained in 2002 showing that Tyr342 is wellpositioned to act as a nucleophile. The significance of Tyr342 was already revealed by that TcTS with either natural or site-directed mutation at this position (Tyr342His, Tyr342Ala, Tyr342Gly, Tyr342Phe, Tyr342Thr) completely lacks trans-sialidase and sialidase activity.60,83-85 However, this idea of Tyr342 acting as a nucleophile was still not adopted due to tyrosines high pKa75 until a chemical trapping study was able to identi fy Tyr342 as the nucleophile in TcTS reaction using an activated substrate.86 2, 3-difluoro sialic acid 7 (Figure 1-13) is an activated substrate since the fluorine atom at C3, adjacent to th e anomeric center, inductively destabilizes the positively charged oxocarbenium ion-like trans ition state reducing both the formation and turnover rates while the fluorid e leaving group at the anomeric center helps the covalent intermediate to be kinetically accessible. When TcTS was incubate d with 2, 3-difluoro sialic acid 7, it was possible to trap and accumulate the cova lent intermediate. Subsequent LC/MS analysis of the peptide digests revealed that the substrate formed a covalent bond to Tyr342, which was later confirmed by X-ray cr ystal structure of this covalent intermediate.77 Thus, in TcTS, Tyr342 indeed acts as a nucleophile coup le with Glu230. It is suggested that there is a charge relay from Glu230 to Tyr342 in order to prevent the Coulombic repulsion of approaching the carboxylate group of the sialic acid directly with a negatively charged nuc leophile. The same procedure was also used to identify Tyr342 as the nucleophi le in TrSA mechanism with subsequent X-ray crystal structure determination of the covalent intermediate.78,87 Comparison of the crystal structure of si alyl-lactose bound TcTS and the covalent intermediate shows that the sialic acid position is fixed from two sides by the strong interactions of its carboxylate group and N-acetyl side chain. However, the anomeric carbon goes through an electrophilic migration changing th e sialic acid ring from the initial distorted skewed-boat

PAGE 41

41 Figure 1-13. Structure of 2,3-difluoro sialic acid us ed for chemical trapping. conformation (B2,5) of the Michaelis complex to a more relaxed chair conformation (2C5) of the covalent intermediate77 (Figure 1-14). This relaxation mi ght also stabilize the covalent intermediate with respect to the Mich aelis complex and lengthen its lifetime. The structural studies clearly indicate Asp59 as the most su itable residue to act as an acid/base catalystto protonate/deprotonate the glycosidic oxygen. Asp59 acting as an acid catalyst for influenza virus sialidase was debate d previously since the pKa value of a solventexposed aspartic acid residue may be too low to act as an acid catalyst.88,89 However, the presence of negatively-charged si alic acid in the cataly tic cleft, and the reduced solvent exposure due to both hydrophobicity of the ca talytic cleft and the aglycone binding were suggested to raise the pKa significantly for Asp59. A recent chemical rescue study that prove using azide ions can restore the trans-sialidase activity of inactive TcTSAsp59Ala mutant forming a sialyl azide product confirmed Asp59 as the acid/base catalyst.90 Our understanding of the mechanism of TcTS has grown over the years as explained in detail in this section and a mechanism shown in Figure 1-14 is now acce pted as the mechanism of TcTS. The relative timing of bond cleavages/ fo rmations and their extent in the transition structure is not clearly depicted in Figure 1-14, leaving it as an open question. The failure of DANA, a molecule structurally similar to an oxocarbenium ion (which is a possible transition 7

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42 Figure 1-14. The proposed mechanism for trans-si alidase catalysis reac tion of TcTS. The donor and acceptor sugar moieties are colored in re d and orange, respectively. In sialidase catalysis reaction of TcTS, a water molecule takes the role of the acceptor lactose.

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43 state), to inhibit TcTS and the KI E results that indicate an associat ive character with little charge development at the anomeric C for the transitio n structure in TcTS provide some information about the transition structure of TcTS mechanis m. These experimental findings will be inquired with computational methods and the details of the mechanism of TcTS and how it differs from the mechanism of TrSA will be i nvestigated in Chapters 3 and 4.

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44 CHAPTER 2 METHODS 2.1. Quantum Mechanics Quantum mechanics calculations take the electro ns into account explicitly and thus, are able to compute the properties of a system that depend on its electronic di stribution. It is also possible to investigate the reactions which involve bond brea king or formation with quantum mechanics. The ab initio and semiempirical quantum mechanics methods as well as the density functional theory will be covered here shortly. The Schrdinger equation lies at the basis of quantum mech anics. The electrostatic potential, V, is taken to be time-independent and thus, the time-i ndependent Schrdinger equation (Equation 2-1) will be discussed in th is section. The wavefunction of a particle, ( r ), defines the state of the particle. In the Schrdi nger equation, the Hamiltonian operator, H,that is given in Equation 2-2acts on ( r ) and returns the wavefunc tion multiplied by a scalar valuecalled the eigenval ueas the result if ( r ) is an eigenstate. The eigenvalue corresponds to the energy of the particular eigenstate. )()( rr E (2-1) V m 22 (2-2) The Schrdinger equation for a one-electron atom can be solved resulti ng in functions that correspond to the atomic orbitals. However, for polyelectronic systems, no exact solution exists for the Schrdinger equation even for He atom since it becomes a three-body problem. Additionally, electron spins have to be accounted for in pol yelectronic systems complicating the solution more. Thus, different approaches ar e taken to obtain approximate solutions for polyelectronic systems. In one approach called the perturbation theory, a similar problem that is

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45 easier to solve than the real one is found and how the differen ce between this problem and the real one will be reflected in the solutions is considered. Born-Oppenheimer approximation which considers the nuclei of the system to be fixed is used to reduce the complexity of the problem of polyelectronic systems. Once the contribution of the nuclei are separated using the Born-Oppenhe imer approximation, the remaining electronic Hamiltonian solely depends on the coordinates of the electrons in the system and its eigenfunctions are called spatial orbitals, ) ( ri For each spatial orbital ) ( ri two different spin orbitalswhich contains information about both the spatial distribution and the electronic spincan be constructed as )()(1 r and )()(2 r in which (w) and (w) represent the spin up and spin down functions. To include the e ffect of electron spins, the exact wave function of the polyelectronic system is re quired to satisfy the antisymmetry principle, which states that the wave functi on must be antisymmetric with re spect to the interchange of the coordinates of any two electr ons, as well as being a soluti on to the Schrdinger equation.91 To satisfy the antisymmetry principle, Slater determinants, an example of which is shown in Equation 2-3 for an N-electron system, are used. k ji k j i k j i k j iN ... )( . )( )( ..... )(..)()( )(..)()( 1 ),...,,( N N N 2 2 2 1 1 1 N21x x x x xx x xx xxx (2-3) The Hartree-Fock approximationequivalent to molecular orbital approximationis one of the most ways to find approximate soluti ons to the electronic Sc hrdinger equation and constitutes a cornerstone for understanding modern chemistry. An antisymmetric wave function for a polyelectronic system can be represented as in Equation 2-3, however, there are no certain forms of spin orbitals to use. Th e variation principle states that the best wave function in such a

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46 functional form is the one which gives the lowest energy, E0, when the full electronic Hamiltonian operator acts on it. Using a procedure to minimize E0 with respect to the spin orbitals chosen, Hartree-Fock equation shown in Equation 2-4 can be derived. In this equation, f(i) is an effective one-electron operator, called the Fock operator, as shown in Equation 2-5 in which vHF(i) is the average potential effective on electron i due to the presence of all other electrons.91 Thus, the Hartree-Fock approximation re duces the problem of the polyelectronic systems to one-electron problems in which each el ectron is assumed to experience an average electronic potential. Since the averag e potential effective on electron i, vHF(i), is dependent on the spin orbitals of all other electrons, the Ha rtree-Fock equation has to be solved iteratively starting from the spin orbitals initially gue ssed until self-consistency is reached and this procedure is called the self consistent field (SCF ) method. The spin orbitals of initial guess are constructed using a finite set of spatial basis functions. ) ()()(i ixxif (2-4) )( 2 1 )(1 2i r Z ifHF M A iA A i (2-5) Semiempirical quantum mechanical methods are based on the Hartree-Fock formalism, however, they use a simpler Hamiltonian than the correct one (e.g. by approximating or totally excluding the two-electron integrals) and use parame ters obtained by fitting to experimental data or the results of ab initio calculations in order to compen sate for using an approximate Hamiltonian. Another approach to the electronic struct ure of polyelectronic systems is density functional theory (DFT). DFT also uses singleelectron functions like Hartree-Fock approach, however, DFT does not calculate the full N-electr on wave function explicitly but only aims to calculate the total electronic energy. DFT is based on the relationship between the overall

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47 electron density and the total electronic ener gy, which can be expressed as that the total electronic energy can be written as a functional form of the overall electron density as in Equation 2-6. In this equation, Vext( r ) is the external potential due to Coulomb interactions with the nuclei and F[ ( r )] is the sum of interelectronic inte ractions and the kinetic energy of the electrons. The variational theory is used to minimize the total electronic energy. Finding the functional forms used for F[ ( r )] constitute one of the difficulti es in this approach. Kohn-Sham equation (Equation 2-7) serves for this purpose and is a sum of the kinetic energy of a system of non-interacting N electrons (with th e same electron density as the re al system), electron-electron Coulombic interaction and exch ange-correlation contributions.92 Self consistent field method is used to solve Kohn-Sham equations for the electrons of the system. )]([)()()]([ rFdrrrVrEext (2-6) N i XC i iEdd d F1 21 21 21 2)]([ )()( 2 1 )() 2 )(()]([ r rr rr rr rr r r (2-7) 2.2. Molecular Mechanics Most of the biological problems are too large to be treated with quantum mechanics. To be able to obtain energies and other properties of such large sy stems, molecular mechanics that computes the energy of a system using onl y the nuclear coordinates (and excluding any information about the electrons) can be used. However, it should be noted that molecular mechanical methods solely can not deal with processes that include bond formation and/or cleavage. To calculate the energy of a system in mo lecular mechanics, force fields which are mathematical functions that empirically describe the contributions of the interactions within a system, like stretching of a bond, changing in a bond angle and dihedral angle, electrostatic

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48 interactions, etc. are used. Current force fields like AMBER,93 CHARMM,94 GROMACS,95 OPLS96 that differ in their functional forms and/ or parametrisation have proved to produce reasonable results in conformational energetics and charge distributions. The mathematical functional form of AMBER force field, which is used throughout this study, is given in Equation 2-8. The first two terms in Equation 2-8 account for energetic penalties associated with the deviation of bonds and angles, respectively, from their equilibrium values. The third term describes how the energy changes as the dihedral angles change, while the last two terms introduce non-bonded (or throughspace) interactions de scribing the van der Waals interactions via a Lennard-J ones potential and the electrosta tic interactions via Coulombs law. atoms ji atoms ji ij ji ij ij ij ij dihedrals n angles eq bonds eq rR qq R B R A n V KrrKRU )( ])cos[1( 2 )()()(612 2 2 (2-8) As well as the functional form of the force fi elds, the parameters (the various constants in the equation) are also important. The parameters of force fields are designed empirically to reproduce various experimental prop erties. Transferability, which is the validity of the same set of parameters for a large group of relevant molecu les, is an important an d useful feature of a force field. Most force fieldsincluding AMBERuse atom types to distinguish an atom with a specific atomic number, hybridization and local environment. Just like quantum mechanical calculations that require the atomic numbers for each atom to be specified, molecular mechanical calculations require the atom type for each atom to be specified. Each parameter in the force field is expressed in terms of atom types, and each parameter necessary for a particular bond or

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49 dihedral angle in a system is supplied from the parameter data files according to the atom types of the constituent atoms. When explicit solvent mol ecules are used to simulate a system, periodic boundary conditions are generally used to exclude the e ffects of interactions of the atoms with the container walls.97 Thus, information on the behavior of the system in bulk solvent, rather than its behavior in a solvent close to a solid surf ace, can be obtained. Using periodic boundary conditions means that a primary cell of volume V which confines all N atoms in the system of interest is imagined to act as a small portion of a bulk material. Basically, the bulk material is imagined to be built by the primary cell surrounded by its exact re plicascalled image cellsin all directions. All cel ls have open boundaries, so atoms can move from one cell to another freely. However, the atom number in each cell is kept constant (N) since for each atom leaving the primary cell, an image of that atom simultaneously enters the primary cell from the opposite face of the cell. During a simulation, only the coordinates of the atoms in the primary cell is stored, and the coordinates of the atom s in the image cells are computed using the appropriate coordinate transformation methods when necessary. 2.3. Hybrid Methods It is currently not feasible to investigate th e reactions of very large systems with quantum mechanics (QM), and molecular mechanics (MM) is insufficient for this purpose since it does not let bonds to be formed or broken. As a solu tion, hybrid methods which combine two or more computational methods in one single calculation are usedfirst realized by Warshel and Levitt98to investigate the reac tions of large systems with high precision. QM/MM methods, which are the most common of hybrid methods, treat the region where the chemistry takes place with quantum mechanics, while the rest of the system is treated with molecular mechanics that has a much less computational cost. The ONIOM method (our O wn N -

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50 layer I ntegrated molecular O rbital molecular Mechanics) implemented in Gaussian03,99 on the other hand, uses an extrapolation approach and is able to combine different methods without having a limitation of two layers at most, and thus is more general. Despite sharing many common properties, QM/M M methods follow different paths in two details. The first detail is how they treat the covalent interaction betw een the quantum mechanics (QM) and molecular mechanics (MM) regions. Once the large system is partitioned into two regions, both regions will have dangling bonds a nd in order to make a quantum mechanical calculation, such dangling bonds have to be saturated. There are se veral methods to resolve this problem; using link atoms,100,101 frozen orbitals,102,103 or pseudopotentials.104,105 Link atoms are used in most QM/MM methodsincluding the one implemented in AMBER9106as well as in the ONIOM method due to their generality. The link atom positions in bot h methods are obtained using a scale factor and the posit ion vectors of the atom that is bonded to the link atom and the atom that is replaced by the link atom. The second detail of QM/MM calculations that differ between various implementations is how the electrostatic interacti on between the two regions is tr eated. In one approach called classical or mechanical embedding, these electrosta tic interactions are calculated between partial point charges of the MM region a nd partial point charges of the QM region as would be in a pure molecular mechanics calculation. For this purpose, partial point charges for each atom in the QM region have to be assigned. In the other appr oach called electronic em bedding, the interaction between charge distribution of the MM region an d the actual charge distribution of the QM region is calculated. This met hod requires inclusion of MM region partial point charges in the QM Hamiltonian and thus, allows the QM region to get polarized according to the MM region charge distribution.

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51 The energy expressions of generic QM/MM a nd ONIOM methods differ from each other. QM/MM methods calculate the tota l energy of the systems as a summation seen in Equation 2-9 (The total energy expressions here are written for mechanical embedding approachwhich is the approach used for the calculations in this studyas will be obvious from that the interactions at the boundary of the two regions are treated with molecular mechanics.). The model system including the link atoms as well is treated with QM methods, and the MM-only system is treated with MM methods. The QM/MM bounda ry interactions are calcul ated including all MM terms that have at least one center in the MM-only region and at leas t one center in the model-only region. MM boundary onlyMMonlyel QMsystemel MMsystem onlyMM MMQM E E E E ,_ mod ,_mod ,_ / (2-9) MMsystemel QMsystemel MMsystem real ONIOM E E E E ,_mod ,_mod ,_ (2-10) On the other hand, the energy expression of ONIOM method given with Equation 2-10 is in the form of an extrapolation and requires three independent calculations.107 The energy of the real system that consists of all atoms is calcul ated at the MM level, and the energy of the model system, which is the focus of interest, is calculated at both QM and MM levels separately. One can infer from Equation 2-10 that most of the MM terms in the model system exist also in the real system and thus, will cancel totally in th e full expression. To prevent a discontinuous potential and to make the result of the energy expression independent of the chosen partitioning of the system, it is suggested to have at least three bonds between the MM region and any bond that will be formed or broken in the reaction. 2.4. Methods for Exploring the Potential Energ y Surface The potential energy is a complicated functi on of the coordinates of all atoms in the system. The variation of energy w ith the coordinates is referred to as the potential energy surface or hypersurface. Visu alization of a potential energy surface is generally not possible

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52 since the potential energy depends on too many va riables (3N coordinates in the case of an Natom-system). Apart from the global minimum of the poten tial energy surface (PES), many other local minima exist on the PES. Each local minimum can be thought as the lowest point in a local energy valley surrounded by higher energy hills. Id entification of minimu m points on PES can be achieved using minimization algorithms. The path that the system follows going from one minimum to another also draws a lot of attention. The highest point on such a pathway between two minima is called a saddle poi nt and the geometry of the system at that point is called a transition structure. Locating the minimu m points on the PES can be used to obtain thermodynamic data, while additional knowledge of the saddle points is necessary to have an insight on kinetics of the system. The minimization problem can mathematically be stated as finding the points on the PES with the first derivatives with respect to each variable being equal to zero and the second derivatives with respect to each variable being positive. Similarly, the saddle points are the points on the PES at which all fi rst derivatives are equal to ze ro and only one of the second derivatives are negative. Although analytical methods exis t to locate the mini ma of a PES, they are not generally used due to the complexity of the form of the potential energy. Instead, numerical methods which gradually lower the energy of the system by changing the coordinates slowly are used. Most of the minimization algorithms go downhill on the PES from the starting point until it reaches the nearest minimum. So far, no al gorithm known can locate the global minimum in a PES starting from an arbitrary point on the PES.92 However, it is also known that the active

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53 structure (for example the active conformation of a drug molecule) is not necessarily the global minimum on the PES or the most highly populated conformation.92 Most minimization methods use derivatives wh ile searching for the minimum. There are different minimization methods that use first de rivatives onlylike steepest descents method and conjugate gradients methodor both fi rst and second derivativeslike the NewtonRaphson method. The first derivative of energy is called the gradie nt, and its direction gives an idea about the relative position of the nearest minimum while its magnitude indicates the steepness of the slope. The gradient is in fact equal to the force, but has an opposite sign. While minimizing the energy of a system, each atom is moved in the opposite di rection to the force acting on it. On the other hand, second derivativ escalled the Hessian matrix when represented in the form of a matrixinform us about the curvature of the system and thus, are used to locate the stationary points of the PES. Generally, minimization methods go downhill in the PES from the starting point by gradually changing the coordinates. The steepest descents method uses a line sear ch or an arbitrary step approach in a direction parallel to the net force in the system.92A line search is finding three points along the line which will satisfy the requirement that the mi ddle point is lower in energy than the other two points. At the minimum point obtained from a line search, the gradient will be orthogonal to the previous direction. Subsequently, following an iterative procedure or using a fitted function decreases the distance between the three pointswh ich will confine the re gion that includes the minimum to a much smaller region. Arbitrary step approach, on the other hand, takes a step of an arbitrary step size in a directi on parallel to the net force by changing the coordinates of the system, and iteratively adjusts the step size chec king whether the energy is lowered in each step. When a step causes an increase in energy in the ar bitrary step approach, the step size is decreased

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54 (e.g. 0.5 of the last step) and thus, leaving the region of the minimum is prevented. The steepest descents method is especially useful at the ini tial part of the minimiza tion process, since it can robustly approach the region of the minimumeven if it is far from the starting structure following the direction of the gradient which re lieves the most unfavorab le features in the starting structure. However, this method follo ws a path that oscillates and continually overcorrects itself, which is generally not an efficient method in the case of a long narrow potential energy well. The conjugate gradients method also has the gradients of all successive steps orthogonal but differs from the steepest descents method in having the direction of successive steps conjugate, rather than orthogonal. The direction of a new step is determined using Equation 2-11 in which vk-1 and vk are successive directions, gk is the gradient and k is a scalar constant. The scalar constant can be calcul ated by different approaches lik e Fletcher-Reeves method (Equation 2-12) or Polak-Ribiere method (E quation 2-13). The line search al gorithm or an arbitrary step approach is also used in the conjugate gradients method. In ge neral, the conjugate gradients method proves to be a much better met hod after the initial strain is removed.92 Thus, the common practice is to use the steepest descents method at the beginn ing of a minimization procedure which is followed by a minimization using the conjugate gradients method. 1 kkk kvgv (2-11) 11 kk kk k.gg .gg (2-12) 11 1)( kk kkk k.gg .ggg (2-13) Apart from the two most popular first deriva tive methods mentioned in the last two paragraphs, the most popular second derivative method, the Newton-Raphson method, will be

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55 described here shortly. For the multidimensi onal potential energy function, the minimum, x*, can be found using Equation 2-14 in which V ( xk) is the force matrix at the point xk. Since the PES is not an ideal quadratic function, an iterative process is necessary to locate the minimum. As seen, Equation 2-14 requires finding the in verse of the Hessian matrix, V ( xk), and this can be computationally too expensive. Thus, Newton-Raphs on method or its variants are generally used for systems of small sizes. Another drawback of Newton-Raphson method is that the minimization can become unstable if the initial point lies in a region for which it is not appropriate to use the harmonic approximation. So another method is generally used to approach the minimum before using the Newton-Raphson method. Many commercial program packages like Gaussian0399 generally use quasi-Newton methods which make use of the vectors representing the current and previous points to update the inverse Hessian matrix instead of constructing it for each iteration. )()(1 k k kxVxVxx* (2-14) Among the most popular first and second derivative minimization methods, first derivative methods are preferred for molecular mechanics calcu lations of large systems due to the high cost of calculating and storing the inverse of the Hessian matrix for such systems. Second derivative methods or their variants are generally used fo r quantum mechanics calculations which can be performed currently for small systems only. While exploring the PES for a system, locating the saddle points as well as distinguishing them from the minima is necessary to understand the kinetics of the syst em. At a minimum point, all eigenvalues are positive, while there is one negative (i.e. imaginary) eigenvalue at a firstorder saddle pointwhich is the saddle point type of interest for our purposes. Most of the times, the conversion of a minimum to another minimum occurs through the change of just one or two

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56 coordinates. Thus, gradually cha nging the relevant coordinates or an appropriate combination of them (called reaction coordinates) and letting the system relax at each step while the reaction coordinates are kept fixed is used to approximately monitor the reaction pathway. Alternatively, the algorithms used for locating the minima can be modified to loca te the saddle points. However, the initial point should be very close to the transition structure for such a method to succeed. In fact, none of the numerical minimization me thods can exactly locat e the minima or the saddle points, but approximate it instead. Thus, the algorithms using these methods have to determine a convergence criterion to stop the mini mization process. For this purpose, either the energy or the coordinates of the atoms of the syst em can be monitored and the calculation can be stopped once there is a smaller difference betwee n the successive steps than a predetermined threshold value. Also the root mean square gradie nt for a system of N atoms can be calculated as shown in Equation 2-15 and be used as a convergence criterion. N RMST3 gg (2-15) 2.5. Molecular Dynamics Simulation Methods Molecular dynam icswhich was first performe d as early as 1957 for a condensed phase system108 and 1971 for liquid water109uses Newtons laws of moti on to generate successive configurations of a system. Solving the diffe rential equations that represent Newtons 2nd law as shown in Equation 2-16, the trajectory of particle s can be obtained. It is possible to solve these equations if the force acting on a particle is eith er zero or constant everywhere independent of the particles position. However, when the force acting on each particle depends on the positions of all other particles in the system, the equati ons become too impossible to solve analytically since the motion of particles are coupled. Thus finite difference method, that breaks down the

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57 integration into small stages each covering an infinitesimal t time window and assumes the force is constant during this time window, is used to integrate the equations of motion. Verlet algorithm110 is the most widely used method to inte grate the equations of motion in molecular dynamics simulations and is summarized in Equa tions 2-17 as an example for such integration algorithms. i z i i y i i x im F dt zd m F dt yd m F dt xdi i i 2 2 2 2 2 2 (2-16) t ttt tt ttttttt ttttttt )()( ) 2 1 ( ...)( 2 1 )()()( ...)( 2 1 )()()(2 2rr v avrr avrr (2-17) An important point before star ting a molecular dynamics simu lation is to decide the time step. A large time step can cause instabilities in the simulation, while a small time step significantly limits the ability to cover the phas e space due to its high cost of computer time. Thus, a compromise between these two factors is necessary while deciding the time step. In simulations of flexible molecules, the time step is suggested to be about 1/10th of the repeat period of the fastest motion of the moleculewhi ch is C-H bond stretching in most biological systems with a period of 10 fs.92 This is why most molecular dynamics simulations either use a time step of 1 fs or freeze the hydrogen stre tchings by constraining those bonds to their equilibrium bond lengths (e.g. SHAKE algorithm111).

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58 The initial coordinates of a system can be obtained from experimental or theoretical modeling studies. In either case, the initial velo cities have to be assigned for each atom which can be done by randomly choosing a velocity for each atom from a uniform distribution, a simple Gaussian distribution or a Maxw ell-Boltzmann distribution at a pa rticular temperature. MaxwellBoltzmann distribution is represented in Equation 2-18 which gives the probabilities for each atom i with a mass of mi to have a velocity vi in the x direction at the simulation temperature T.92 The initial velocities are then generally adjusted to provide an overall momentum of zero for the system. ) 2 exp() 2 ()(2 2/1Tk vm Tk m vPB ixi B i ix (2-18) Prior to the production phase molecular dynamics simulation, the system is relaxed to reach a stable configuration. Various properties of the system especially its energy, are monitored during this phase until they reach stable values. For the simulations of proteins, it is necessary to constrain the protei n backboneor the entire protein in the case of explicit solvent MD simulationsat the beginning of the relaxation phase to conser ve its secondary and tertiary structures. These constraints are then gradually reduced and finally released during the relaxation phase.

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59 CHAPTER 3 QUANTUM MECHANICS/MOLECULAR MECH ANICS STUDY OF THE CATALYTIC MECHANISM OF TRYPANOSOMA cruzi TRANS-SIALIDASE AND TRYPANOSOMA rangeli SIALIDASE USING ONIOM 3.1. Introduction Trypanosoma cruzi trans-sialidase (TcTS) specifica lly catalyzes the transfer of -(2-3) sialic acid from sialoglycoconjugates to -galactosyl glycoconjugates, retaining the anomeric configuration, while Trypanosoma rangeli sialidase (TrSA) is respons ible only for the hydrolysis of -(2-3) glycoside bonds. TcTS is found to act through a ping-pong mechanism (Figure 1-14) and shares with TrSA the first step in whic h the sialic acid is scavenged from the donor glycoconjugate with some nucleoph ilic participation of the en zyme. Whether this nucleophilic participation ends up collapsing into a covalent intermediate or remains as an oxocarbenium ion intermediate ( 2 in Figure 1-6) in each mechanism has been long discussed for enzymes of the similar families, even for the lysozyme.112-114 Strong nucleophilic partic ipation in the transition structure of TcTS revealed by KIE studies pointed towards subs equent covalent intermediate formation.83,115 Quenching the catalytic reaction of a radioisotope -labeled ligand clearly indicated that TcTS mechanism involved a cova lent bond formation between the ligand and the enzyme.83,115 A separate effort later achieved trapping and identifying the covalent intermediate of TcTS and TrSA using 2-deoxy-2,3difluorosialic acid as the ligand ( 7 in Figure 1-13).77,78,86 Despite elucidating the identity of the unusual nucleophile of a Ty r/Glu couple, there is still a possibility that activated fluoro-ligands follow a different reaction path than the natural ligand. This possibility should especially be investigated for TrSA for which there is no direct evidence for the natural substrate forming a covalent intermediate and the covalent intermediate formation might be the result of a modified mechanism forced by the activated substrate. The short lifetime of the covalent intermediate of TrSA revealed by the kinetic studies of the covalent

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60 intermediate78 may also be an indicator of a finely-t uned mechanism which has just shifted from a strong nucleophilic partic ipation to a covalent intermediate formation due to the effect of fluorine substituents of the substrate. Since no difference in the mechanisms of TcTS and TrSA could be elucidated with the available experimental studies so far and DANA, an oxocarbenium-like molecule and a very efficient inhibitor for TrSA, is unable to inhibit TcTS44,72 possibly pointing to different transition states, we decided to investigat e the mechanisms of the two enzy mes computationally in detail. Is there a covalent intermediate formation for both TcTS and TrSA reactions with the natural substrates actually? Is the catalysis an SN1-like or an SN2-like mechanism in each case? How do the energetic barriers compare to each other? Can the energetic barriers explain why TcTS prefers transferring the sialic acid to the glycoc onjugate rather than to a water molecule? Why can TrSA only catalyze hydrolysis? The answers to these questions can shed light to our understanding of the mechanism and can serve as a guide for future inhbitor design studies. First, we investigated the covalent intermediate formation step (Figure 3-1) in TcTS and TrSA. For this purpose, we prepared potential energy surfaces (PES) using possible reaction coordinatesbond lengthsfor both TcTS and TrSA mechanisms. Our study is presented here in a chronological order. At the time this study began, the KIE studies had just shown covalent intermediate formation in TcTS and had anticipated Tyr342 to be the nucleophile,52,83 as well as the first X-ray crystal structures of TcTS were just obtained.75 However, there was still a lot being debated about the mechanism by several e xperimental groups, even about whether Tyr342 could behave as a nucleophile or not.75 At the beginning, we assumed an SN2-like reaction and used RC1 and RC2 (Figure 3-2) as our reac tion coordinates. We were also limited by Gaussian0399 not allowing the use of combination of two or more bonds as a reaction coordinate.

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61 However, the results from these 2-dimensional SN2-like PESs as well as the results from hybrid calculations performed in AMBER10 that will be presented in the next chapter indicated the use of RC3 and RC4 would be a bett er choice. Thus, we performed 1-dimensional potential energy scans using RC3. To compare with the trans-sial diase reaction, we have also investigated the energetics of the sialidase reaction of TcTS using an RC3 scan Since the sialidase re action differs from the trans-sialidase reaction only in the step after the covalent intermediate formation, only the differing step is studied fo r the sialidase reaction. 3.2. Methods 3.2.1. Relaxation of the TcTS and TrSA Systems The X-ray crystal structure of TcTS in com plex with sialyllactose ( www.rcsb.org pdbID: 1S0I) is used as a starting point to investig ate Tyr342 attack to the sialyllactose in TcTS. Everything except for the first 371 residues from chain A and sialyllact ose is deleted. The mutated residue Ala59 is mutated back to an aspartic acid. The resulting pdb is fed into the Xleap module of AMBER9 molecular dynamics package93,116,117 where the necessary H atoms and any other missing atoms are added as well as being solvated in a truncated octahedral cell of TIP3P118 explicit water molecules. FF99SB119,120 and Glycam04121-123 force fields are used to construct the topology files. Sande r module of AMBER9 is used fi rst to minimize the energy of the system and then to relax it under periodic boundary conditions with a procedure explained in detail as follows: (1) 1000 steps of minimization with the protein and the ligand fixed, (2) 2500 steps of minimization, (3) 20 ps of molecular dynamics at constant volume with the protein and the ligand weakly restrained while increasing temper ature from 0 K to 300 K with Langevin dynamics,124 (4) 100 ps of molecular dynamics at constant pressure while keeping temperature at 300 K with Langevin dynamics,

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62 (5) 3.9 ns of molecular dynamics at constant pressure while keeping temperature at 300 K with Langevin dynamics. Throughout all minimizations and molecular dynam ics, the cutoff used for nonbonded distances is 10 All molecular dynamics is performed using SHAKE algorithm for H-involving bonds. Since there is no X-ray crystal structure ava ilable for sialyllactos e-bound form of TrSA, the X-ray crystal structure of TrSA in complex with 2-deoxy-2, 3-dehyd ro-N-acetyl neuraminic acid (DANA) ( www.rcsb.org pdbID: 1N1T) is used as a star ting point to investigate Tyr342 attack to the sialylla ctose in TrSA. Only the first 375 re sidues and DANA are conserved. The sialic acid part of the sialyl lactose in 1S0I.pdb is superimpos ed with DANA in 1N1T.pdb and the appropriate coordinates for sialy llactose for TrSA are constructed in this way. The resulting pdb is completed for the missing H atoms and solvated as stated above for TcTS by the Xleap module. Minimization and relaxation of the system with the same procedure as in the TcTS case followed. The X-ray crystal structure of TcTS covalent intermediate ( www.rcsb.org pdbID: 2AH2) is used as a starting point to investigate water attack to TcTS covalent intermediate. Everything except for the first 371 residues and sialic acid is deleted. The resu lting pdb is completed for the missing H atoms and solvated as stated above for sialyllactose-bound TcTS by the Leap module of AMBER9. Minimization and relaxation of the system with the same procedure as in the sialyllactose-bound TcTS case followed. 3.2.2. Preparation of the TcTS and TrSA Model Systems The relaxed geom etry of TcTS with sialyllact ose is used for model system preparation of TcTS to use in trans-sialidase reaction simulation. Sialyllactose, Trp312 and all residues that are within 3 of sialic acidthe ligand, 17 protei n residues and 4 water moleculesare chosen to

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63 constitute the model system. A hydrogen atom at the N-end and an OH group at the C-end are added for each residue for completeness. Total number of atoms in the model system is 464. The relaxed geometry of TrSA with sialyllact ose is used for the model system preparation of TrSA. Sialyllactose an d all residues within 3 of sialic acidthe ligand, 14 protein residues and 13 water moleculesare chosen to constitu te the model system. The N-end and C-end of included residues are completed with an H at om and an OH group as well. Total number of atoms in the model system is 420. The relaxed geometry of TcTS covalent interm ediate is used for model system preparation of TcTS to use in sialidase r eaction simulation. Sialic acid, Tr p312, Tyr119 and all residues that are within 3 of sialic acidt he ligand, 16 protein residues a nd 8 water moleculesare chosen to constitute the model system. A hydrogen atom at the N-end and an OH group at the C-end are added for each residue for completeness. Total number of atoms in the model system is 404. 3.2.3. ONIOM Input Setup For ONIOM calculations of sialyl lactose-bound TcTS using Gaussian03,99 the quantum region is chosen to consist of Asp59, Glu230, Tyr 342 and the sialyllactose, a total of 139 atoms. All backbone atoms except for C atoms of Tyr342, Glu230 and Asp59 are frozen. All other atoms including the entire sialyllactose and wate r molecules are free. Only one link atom is necessary at the N-end of Glu230. The charge of the whole system and the charge of the quantum region are +2 and -2, respectively. The initial ONIOM calculations used Hartree-Fock level of theory with a 3-21G basis set as the high level of theory and AMBER force field for the low level of theory. The quantum region for sialyllactose-bound TrSA is chosen to consist of Asp59, Glu230, Tyr342 and sialyllactose, a total of 139 atoms. All backbone atoms except for C atoms of Asp59, Glu230 and Tyr342 are frozen. The charge of the whole system and the charge of the

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64 quantum region are +1 and -2, respectively. The initial ONIOM calculations for TrSA are performed with the same levels of theory as those used for TcTS. For ONIOM calculations of TcTS c ovalent intermediate using Gaussian03,99 the quantum region is chosen to consist of Asp59, Glu230, Tyr342 and the sia lic acid, a total of 139 atoms. All backbone atoms except for C atoms of Tyr342, Glu230 and Asp59 are frozen. All other atoms including the entire sialic acid and water molecules are free. Only one link atom is necessary at the N-end of Glu230. The charge of the whole system and the charge of the quantum region are +1 and -2, respectively. The initial ONIOM calculations used Hartree-Fock level of theory with a 3-21G basis set as the high level of theory and AMBER force field for the low level of theory. 3.2.4. Potential Energy Surface Scan Setup 3.2.4.1. Simulating the trans-sialidase reaction of TcTS in a SN2 way A 2-dimensional potential energy surface (PES) for the covalent intermediate formation step (Figure 3-1) is prepared for TcTS by scanning the two distances: (i) The distance between the anomeric C of sialic acid and the O atom of Tyr342 hydroxyl group (RC1 in Figure 3-2), (ii) The distance between the O atom of Glu230 carboxylate group and the H atom of Tyr342 hydroxyl group (RC2 in Figure 3-2). The attack distance is scanned between values of 1.46 and 3.46 with increments of 0.25 and the H transfer distance is scanne d between values of 0.96 and 1.76 with increments of 0.2 In order to get more pr ecise values, the initial PES is reconstructed by gradually increasing the high level of theory in ONIOM calculations first to HF/6-31G* and then to B3LYP/6-31G* using the output of each level of theory as an input for the subsequent higher level of theory. A final PES is constructed by enlarging the quantum region to include the

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65 arginine triad (Arg35, Arg245 a nd Arg314) that results in 220 atoms in the quantum region. Figure 3-1. The covalent intermediate formation step 3.2.4.2. Simulating the trans-sialidase reaction of TrSA in a SN2 way For TrSA, a similar 2-dimensional PES as in the TcTS case is prepared by scanning the same two distancesRC1 and RC2. The attack di stance is scanned between values of 1.42 and 3.42 with increments of 0.25 and the H tr ansfer distance is scanned between values of 0.96 and 1.96 with increments of 0.2 The same procedure of consecutive PES calculations by increasing the level of theory fo r TcTS is followed for TrSA. The final quantum region consists of Asp59, Glu230, Tyr342 and si alyllactose and the Arg triad (Arg35, Arg245 and Arg314). 3.2.4.3. Simulating the trans-sialidase reaction of TcTS in a SN1 way A 1-dimensional potential energy surface (PES ) is prepared for TcTS by scanning the distance between the anomeric C of the sialic acid and the glycosidic O of lactose (RC3 in Figure 3-2). The starting geometry is the same as the one used for investigating the SN2 mechanism. The attack distance RC3 is scanned between values of 1.45 and 3.85 with increments of 0.20 The PES is constructed at the level of B3LYP with 6-31G* basis set and using a large quantum

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66 region including the Arg triad (only the guanidinium regions of Arg35, Arg245 and Arg314 this time) as well as Asp59, Glu230, Tyr342 and sialyllactose (the 2nd and 3rd C atoms on the glycerol side chain of sialic acid and their substituents are excluded from the quantum region as well as the entire glucose and uppe r half of the galactose). Figure 3-2. Reaction coordinates us ed to generate poten tial energy surfaces. Th e sialic acid and lactose parts of sialyllactose are colo red in black and orange, respectively.

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67 Since the RC3 scans attempting to simulate SN1 reaction above ended up in oxocarbenium ion formation, a subsequent RC1 scan is perfor med to see the energetic profile of covalent intermediate formation from the oxocarbenium i on. Starting from the optimized structure of RC3=3.05 in the very first RC3 scan above, we prepared a 1-dimensional PES by scanning the RC1 distance from 2.76 to 1.46 with increments of 0.10 3.2.4.4. Simulating the si alidase reaction of TcTS The trans-sialidase catalysis and sialidase/hydr olysis catalysis only di ffer in the steps after the cova lent intermediate formation. The attack of a water molecule to the covalent intermediate is also simulated by gradually pushing the nearest water molecule towards the anomeric C in the TcTS covalent intermediate until the sialic acid formsfrom 3.46 to 1.46 The two minima in the potential energy scans are optimized releas ing all constraints and the resulting geometries of these unconstrained optimizations are confirme d to be minima by the all-positive frequencies computed for them. The PES is constructed at the level of B3LYP with 6-31G* basis set and using a quantum region including Asp59, Glu230, Tyr342 and the sialic acidbound to Tyr342. 3.3. Results and Discussion 3.3.1. The SN2-like trans-Sialidase Reaction of TcTS For TcTS and TrSA, the first step of the reac tion, which is scavenging the sialic acid from the lactose, has been the subject of discussions about whether th e nucleophilic participation from Tyr342 ends up collapsing into a co valent intermediate or remain s as a stable oxocarbenium ion intermediate. Since computational methods allow us to work with the natural ligand by easily changing the F atom used to obtai n X-ray crystals to an H atom, we investigated the detailed mechanism of TcTS as well as of TrSA. For this purpose, we obtained the potential energy surfaces (PES) of an SN2-like reaction by constraining two cr itical bond distances for TcTS and TrSA:

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68 (i) RC1, the distance between Tyr342 hydroxyl O a nd the anomeric C of sialic acid, (ii) RC2, the distance between Tyr 342 hydroxyl H and Glu230 carboxylate O. The PES of TcTS (Figure 3-3) shows two mi nima, one that corres ponds to the Michaelis complex and another to the covalent intermediate. The covalent intermediate minimum (RC1=1.46 RC2=0.96 ) is only 0.21 kcal/ mo l higher in energy than the Michaelis complex minimum (RC1= 3.21 RC2= 1.16 ), showing that the covalent intermediate is energetically accessible. Ideally, one would do a frequency calculation to characterize a stationary state, however, it was not possible to do it with such a large system in ONIOM. Thus, unconstrained optimizations starting from the best local mini mum candidate are used to reach the closest minimum and to show that we are indeed at a stable local minimum. The covalent bond formation is confirmed by totally unconstrained optimization of the covalent intermediate structure in the TcTS PES, which resulted in a structure with RC1=1.50 and RC2=0.99 and an energy that is 0.56 kcal/ mol lower than the TcTS Michaelis complex. Being 1.50 long, the new covalent bond formed is a little longer than that of the X-ray crystal structure (1.42 ).77 The calculations of Bottoni et al.125 also showed a similar C-O bond distance of 1.522 for the covalent bond in a glycosyl-enzym e intermediate of lysozyme. Additionally, the structure that corresponds to the TcTS Michaelis complex (RC1= 3.21 RC2= 1.16 ) shows a strong hydrogen bond between Tyr342 hydroxyl group and Glu230 carboxylate group. We also observe that the hyd rogen transfer between Tyr342 and Glu230 can occur easily in TcTS with a low energy cost of 2.47 kcal/ mol despite the high pKa difference of tyrosine and glutamic acid residues and 4, respectively. To show the effect of the enzyme environment, we have calculated th e energetic difference between (Tyr.O+ Glu.H) and (Tyr.OH + Glu-)which just differ in the H atom positionusing the exact same coordinates for these two residues from the QM/MM calculations including the entire active site. The difference is

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69 found to be 9.97 kcal/ mol if only the tyrosine and glutamic acid residues are included in the calculation, and it rises to 16.18 kcal/ mol if the sialic acid is included as well as the two residues. These calculations show that the enzyme environment indeed significantly lowers the energetic barrier of a rather unfavor able hydrogen transfer reaction. Analyzing the structures that correspond to the in dividual points on TcTS PES presents clues about the detailed mechanism of the reaction. The top left plot in Figure 3-4 indicates that lactose conserves its bond to the sialic acid (a ~1.45 long C-O bond) in the Michaelis complex until the bond breaks at RC1=2.21 As RC1 is forced to become shorter, lactose keeps moving further away from the sialic acid In short, lactose breaks its bond to the sialic acid much before that Tyr342 forms a covalent bond to th e sialic acid, which happens at RC1 1.46 Figure 3-3. Potential ener gy surface of TcTS for SN2-like covalent intermediate formation step. Energies are in kcal/ mol. RC1 RC2 Energy Covalent intermediate Michaelis complex

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70 While breaking its bond to the sialic acid, the lactose molecule simultaneously transfers a H atom from the Asp59 carboxylic aci d group. This is shown clearl y in the top right plot in Figure 3-4 as the distance of the glycosidic O to H atom in Asp59 carboxylic acid decreases abruptly from 2.32 at RC1=2.46 to 0.98 at RC1=2.21 Figure 3-4. Changes in geometric pr operties of sialic acid during SN2-like covalent intermediate formation of TcTS. Pyramidalization is m onitored by the dihedral angle of C3-C1O5-C2 in sialic acid which becomes 0 for a completely planar oxocarbenium ion. Distances are in and dihedr al angles are in degrees.

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71 The pyramidalization around the anomeric C of the sialic acid is another important parameter to monitor since the planarity around the anomeric C is a sign of oxocarbenium ion formation. The bottom right plot in Figure 3-4 shows that the pyramidalization values are all negative for the region where RC1 is 2.46 or larger and all positive for the region where RC1 is 2.21 or smaller; that is, a change in the direction of the apex of the pyramid with the anomeric C as the apex has occurred. Thus, the planar oxocarbenium form of sialic acid must be forming in the region of 2.21 < RC1 <2.46 Accordingly, in this region, the bond between the anomeric C and its neighboring O5 atom in the sialic acid ring shortens as a result of the electron donation from the lone pa ir of O5 atom to the empty pz orbital of the positively charged anomeric C (bottom left plot in Figure 3-4). This C-O bond becomes shortest (1.27 ) at the point of RC1=2.21 and RC2=1.76 In summary, this PES shows that the covalent intermediate formation is energetically plausible with a covalent intermediate almost equienergetic with the Michaelis complex. The observation of that the leaving grouplactosebreaks its bond to th e sialic acid long before Tyr342 forms a covalent bond to the anomeric carbon and there is a metastable oxocarbeniumlike intermediate forming before the covalent in termediate (in the valley right next to the covalent intermediate in Figure 3-3) led us to think that an SN1-like mechanism is more likely for this step in TcTS. Thus, we performed 1-dimens ional potential energy scans of RC3 (instead of RC1/RC2) to investigate the covalent inte rmediate formation mechanism further. 3.3.2. The SN2-like trans-Sialidas e Reaction of TrSA The PES of TrSA (Figure 3-5) also has tw o minima that correspond to the Michaelis complex and the covalent intermediate. The covalent intermediate minimum (RC1=1.67, RC2=0.96 ) is 4.44 kcal/mol higher in energy than the Michaelis complex minimum (RC1=3.42 RC2=1.76 ) and is energetica lly accessible. The covalent bond formation is

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72 confirmed by totally unconstrained optimization starting from the covalent intermediate structure at RC1=1.46 and RC2=0.96 which resulted in a structure with RC1=1.50 and RC2=0.97 and an energy that is 1.09 kcal/ mol hi gher than the TrSA Michaelis complex. The structure that corresponds to the TrSA Michaelis complex (RC1=3.42 RC2=1.76 ) also has H-bonding between Tyr342 hydroxyl group and Glu230 carboxyl ate group, but not as strong as in the TcTS case. The hydrogen tr ansfer between Tyr342 and Glu230 is much more difficult in TrSA with an energy barrier of 16 kcal/ mol. When the H is located on Glu230 instead of Tyr342, the structure is about 10 kcal / mol higher in energy co mpared to the case in which H is retained on Tyr342. Favoring H to reside on Tyr342, but not on Glu230 for the Michaelis complex, TrSA differs from TcTS. Figure 3-5. The potential energy surface of TrSA for SN2-like covalent intermediate formation step. Energies are in kcal/ mol. Covalent intermediate Michaelis complex

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73 The top left plot in Figure 36 shows that lactose to sialic acid bond is conserved in the TrSA Michaelis complex until the bond is broken at RC1=1.92 Similar to the TcTS case, lactose slowly moves away from the sialic acid as RC1 becomes shorter. However, TrSA differs from TcTS in requiring shorter RC1 values compared to TcTS to break the lactose to sialic acid bondthe bond was broken at RC1=2.21 in TcTS. This can clue a more SN2-like mechanism for TrSA. The simultaneous hydrogen transfer fro m Asp59 carboxylic acid group to lactose is realized here, too, as seen in the top right plot in Figure 3-6. The pyramidalization values are all negative fo r RC1 of 2.17 and larger values and all positive for RC1 of 1.92 and shorter values (bo ttom right plot in Figure 3-6). These data point that the planar oxocarbenium form of sialic acid forms in the region 1.92 < RC1 < 2.17 This is also supported by the shortening of the bond between anomeric C and neighbouring O5 atom in this region, as seen in Figure 3-6. 3.3.3. The SN1-like trans-Sialidas e Reaction of TcTS To investigate the SN1-like mechanism, the bond distance between the anomeric C and the glycosidic O (RC3 in Figure 3-2) is scanned. This potential energy scan (Table 3-1) showed a stable oxocarbenium ion formation with an ener gy barrier of about 31 kcal/ mol (and an energy barrier of 22 kcal/ mol for the reverse reaction). Even when lactose is pulled further away from the oxocarbenium ion form of the sialic acid, no simultaneous attack of Tyr342 to the anomeric carbon is observed. The two minima are also c onfirmed by totally unconst rained optimizations which gave almost exactly the same geometries as those of the starting st ructures and with allpositive frequencies. As the oxocarbenium i on forms, the hydroxyl O atom of Tyr342 approaches the anomeric C and gets more polariz ed. This nucleophilic assistance is also reflected in the decrease of anomeric C charge as Tyr342 approaches.

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74 Figure 3-6. Changes in geometric pr operties of sialic acid during SN2-like covalent intermediate formation of TrSA. Pyramidalization is m onitored by the dihedral angle of C3-C1O5-C2 in sialic acid which becomes 0 for a completely planar oxocarbenium ion. Distances are in and dihedr al angles are in degrees. Following the RC3 scan that facilitated the fo rmation of the oxocarbenium ion form of sialic acid, an RC1 scan is performed to simulate the conversion of the oxocarbenium ion to the covalent intermediate. For this purpose, the optimized geometry at RC3=3.05 (in which RC1=2.76 ) is used as a starting geometry and Tyr342 hydroxyl group is pushed first to RC1=2.36 and then gradually closer to the anom eric C with increments of 0.10 Table 3-2

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75 shows that the energy barrier for oxocarbenium ion to form the covalent intermediate is very low (5 kcal/mol). Also, the covalent intermediate obt ained from this RC1 scan is optimized without any constraints showing its stabilit y and the final covalent intermed iate structure is proved to be a minimum by its all-positive frequencies calculated. Table 3-1. Results of RC3 scan for TcTS that show stable oxocarbenium ion formation RC3 () Energy (Hartree) Relative energy (kcal/mol) RC4 () RC1 () RC2 () Pyramidalization around anomeric C () Charge on the anomeric C Charge on Tyr342 hydroxyl O 1.45 -3631.020164 0.00 2.49 3.80 1.68 -33.0 0.665 -0.863 1.65 -3631.010161 6.28 2.48 3.60 1.65 -28.4 1.85 -3630.994516 16.09 2.50 3.42 1.63 -23.6 0.628 -0.875 2.05 -3630.980832 24.68 2.53 3.25 1.61 -18.2 0.618 -0.885 2.25 -3630.970787 30.98 2.57 3.11 1.59 -13.4 0.619 -0.896 2.45 -3630.974628 28.57 2.32 2.85 1.57 -8.2 0.634 -0.908 2.65 -3630.995837 15.27 1.03 2.84 1.61 -5.2 0.607 -0.906 2.85 -3630.994196 7.55 1.03 2.79 1.62 -3.1 3.05 -3631.006762 8.41 1.03 2.76 1.63 -1.4 0.592 -0.907 3.25 -3631.006251 8.73 1.03 2.74 1.65 0.5 0.591 -0.906 3.45 -3631.006098 8.83 1.03 2.70 1.65 1.9 0.596 -0.907 3.65 -3631.005888 8.96 1.03 2.83 1.68 1.0 0.585 -0.900 3.85 -3631.005800 9.01 1.00 3.00 1.70 2.0 0.579 -0.888 1.45 nor -3631.020164 0.00 2.49 3.80 1.68 -33.0 3.85 nor -3631.005798 9.01 1.00 3.00 1.70 1.9 nor = optimization with restraints released. Charge values are Mulliken charges. One can easily imagine that for the sialic acid transfer reaction to be completed the exact reverse reactions have to occur as seen in Figur e 3-7 if lactose acts both as the sialic acid acceptor and donor. Thus, with the potential ener gy profiles obtained, the potential energy diagram for the complete trans-sialidase reactio n can be built as seen in Figure 3-8. 3.3.4. The Sialidase Reaction of TcTS The trans-sialidase catalysis and sialidase/hydrol ysis catalysis only diffe r in the steps after the cova lent intermediate formation. To be able to compare the trans-sialidase catalysis with sialidase catalysis energetically, th e attack of a water molecule to the covalent intermediate is also simulated. For this purpose, the nearest water molecule is gradually pushed to the anomeric

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76 Figure 3-7. The mechanism proposed for trans-si alidase catalysis reaction of TcTS. The donor and acceptor sugar moieties are colored in re d and orange, respectively. In sialidase catalysis reaction of TcTS, a water molecule will take the role of the acceptor lactose.

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77 Table 3-2. Results of RC1 scan for TcTS that show conversion of the oxocarbenium ion to the covalent intermediate. RC1 () Energy (Hartree) Relative energy (kcal/ mol) RC2 () RC3 () RC4 () Pyramidalization around An.C () 2.36 -3631.003782 10.28 1.61 3.13 1.03 6.0 2.26 -3631.002332 11.19 1.61 3.38 1.03 10.2 2.16 -3631.000186 12.54 1.58 3.40 1.03 12.5 2.06 -3630.997624 14.14 1.55 3.42 1.03 15.2 1.96 -3630.997617 14.15 1.47 3.44 1.03 18.0 1.86 -3631.002469 11.10 1.01 3.49 1.03 22.8 1.76 -3631.004610 9.76 1.00 3.56 1.03 25.8 1.66 -3631.007052 8.23 0.99 3.62 1.03 28.4 1.56 -3631.009020 6.99 0.99 3.68 1.03 31.0 1.46 -3631.015184 3.12 0.99 4.07 1.03 34.3 1.46 nor -3631.015929 2.65 0.99 4.05 1.03 33.0 nor = optimization with restraints released. C of the TcTS covalent intermediate until the sia lic acid forms (Table 3-3). The three minima in this potential energy profile are optimized releasing all constraints and the resulting geometries of these unconstrained optimizations are confir med to be the minima of this reaction by frequency calculations (Table 3-3). Combining the potential energy profiles obtained, the comple te potential energy diagram of the sialidase reaction of TcTS is constructed as seen in Fi gure 3-9. Comparing Figure 3-8 and Figure 3-9 shows us the importance of covalent intermediate formation. The potential energy barrier for water attacking the c ovalent intermediate is higher th an the barrier for the acceptor lactose attacking. If the covalent intermedia te did not form and the reaction was proceeding directly through the oxocarbenium ion form of sialic acid inst ead, the potential energy barriers would favor the water attack ra ther than the lactose attack. So the potential energy diagrams obtained in this study are in line with the experimental results showi ng that trans-sialidase catalysis is more efficient in the presence of acceptor sugar molecu les and only sialidase catalysis is observed in the absence of acceptor sugar molecules (Table 1-1).

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78 Figure 3-8. Potential energy diag ram calculated for trans-sialidas e catalysis reaction of TcTS. Table 3-3. Results of RC3 scan that show a water molecule attacking the covalent intermediate in TcTS. RC3 () Energy (Hartree) Relative energy (kcal/mol) RC4 () RC1 () RC2 () Pyramidalization around anomeric C ( ) 3.46 -2857.099947 0.00 1.88 1.51 0.99 30.0 3.21 -2857.096624 2.09 1.85 1.52 0.99 28.8 2.96 -2857.089697 6.43 1.84 1.54 0.99 27.0 2.71 -2857.080102 12.45 1.84 1.57 0.99 24.4 2.56 -2857.071826 17.65 1.83 1.60 0.99 22.3 2.21 -2857.076973 14.42 1.63 2.70 1.57 -7.9 1.96 -2857.067301 20.49 1.56 2.90 1.54 -16.6 1.71 -2857.071060 18.13 1.40 3.06 1.53 -23.3 1.46 -2857.091428 5.35 0.99 3.22 1.53 -31.1 3.46 nor. -2857.100446 -0.31 1.89 1.50 0.99 30.5 2.21 nor. -2857.085089 9.32 1.77 2.49 1.52 5.4 1.46 nor. -2857.091438 5.34 0.99 3.22 1.54 -31.2 nor = optimization with restraints released.

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79 Figure 3-9. Potential energy di agram calculated for sialidase catalysis reaction of TcTS. 3.4. Conclusion The results obtained by the calculations with ONIOM method confirm the covalent intermediate formation for both TcTS and TrSA. In both SN2-like and SN1-like approaches, the covalent intermediate is found to be almost equienergetic with the Michaelis complex. For TcTS, the trans-sialidase and sia lidase catalysis reactions are also compared preparing their potential ener gy diagrams which pointed out the importance of covalent intermediate formation for trans-sialidase cataly sis. The potential energy diagrams also showed that if the reaction proceeded directly through ox ocarbenium ion, sialid ase catalysis would be favored rather than trans-sialidase catalysis in TcTS even in the presence of an acceptor molecule. In the next chapter, a similar study using QM/MM method in AMBER10 will be presented.

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80 CHAPTER 4 QUANTUM MECHANICS/MOLECULAR MECH ANICS STUDY OF THE CATALYTIC MECHANISM OF TRYPANOSOMA cruzi TRANS-SIALIDASE USING AMBER 4.1. Introduction In order to check whether the results pr esented in Chapter 3 are dependent on the QM/MM method used, a sim ilar study on the mechanism of Trypanosoma cruzi trans-sialidase is done using a different QM/MM met hodthe QM/MM method in AMBER10.117 Constrained minimizations using the same reaction coordi nates are performed fo r this purpose. 4.2. Methods 4.2.1. Model Preparation The starting structures fo r simulations are prep ared from the sialyl-l actose (sialic acid in TrSA case) bound high-resolution crystal structures of TcTS and TrSA ( www.rcsb.org pdbID: 1S0I & 1N1T). Only the catalytic domains of the proteinsthe first 371 residues in TcTS and the first 375 residues in TrSAare included fo r the models. All the crystallographic water molecules are deleted and lactose is replaced by a methoxy group bound at the anomeric C atom of the sialic acid using the LEA P module which also adds the hydrogen atoms at pH 7. However, the protonation states of the three active site residues, Tyr342, Glu230 and Asp59 (numbered as in TcTS), are adjusted in accord to the propos ed mechanisms and will be mentioned for each case. Then, the systems are solvated in a truncated-octahedron wa ter box of TIP3P water molecules. Parameters from FF99SB120,126 and Glycam04121-123 force fields are used. The systems are relaxed using the following procedure in AMBER9 suite: (1) Minimization for 1000 stepsthe first 10 steps using the steepest descents method and the rest using the conjugate gradients methodfixing the positions of all the main chain atoms except for H and carboxylic O atoms. The cutoff used to truncate nonbonded pairs is 8.0 (2) Molecular dynamics simulation for 1.5 picoseconds at 300 K at constant pressure with SHAKE used for fixing bonds that involve H atoms. (3) Minimization for 1000 more st eps just as in step 1.

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81 (4) Minimization for 1000 more steps as in step 1 except with much weaker constraints on the main chain atoms. (5) Minimization for 1000 more steps as in step 1 with all constraints on atoms released. (6) Molecular dynamics simulation of 5 pi coseconds as in step 2 except that the constraints on the atoms are 1/5th of the initial value. (7) Molecular dynamics simulation of 15 picoseconds as in step 2 except that the constraints on the atoms are 1/10th of the initial value. (8) Molecular dynamics simulation of 5 pi coseconds as in step 2 except that the constraints on the atoms are all released. (9) Molecular dynamics simulation of 0.5 nanoseconds as in step 8. 4.2.2. Initial Minimization All water molecules in the relaxed model system are deleted a nd a nonperiodic, vacuum model of TcTS system is simulated. 5000 steps of pure MM minimization for the entire system are followed by 5000 steps of QM/MM minimization. The initial pure MM minimization is performed by 10 cycles of steepest descent me thod followed by conjugate gradients method for the rest of the minimization with a 1000 cutoff for nonbonded interactions at 100 Kprovided by a Langevin thermostat with 2 ps-1 collision frequency. Th e QM/MM minimization is performed with the same procedure as the pure MM minimization. PDDG/PM3,127 a semiempirical model, is used as the QM met hod and the QM region consists of Tyr342, Glu230, Asp59 and the ligand. The convergence criteri on used for the SCF calculation is 10-4 kcal/ mol and for the root mean square of the energy gradient is 10-4 kcal/ mol 4.2.3. QM/MM Potential Energy Surface Preparation The potential energy su rfaces (PES) of sialyl -transfer reaction of TcTS is prepared by constrained minimizations using QM/MM calcul ations of Amber10 suit. PDDG/PM3 method is used as the QM method and the QM region consists of Tyr342, Glu230, Asp59 and the ligand. The same procedure as the initial QM/MM minimization in vacuum is followed for 1000 steps except that the convergence criteri on for the root mean square of the energy gradient is 10-2 kcal/

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82 mol this time and a harmonic biasing potenti al with a force constant of 500 kcal/ mol 2 is used. First, an SN2-like mechanism is simulated using RC1 and RC2 as the reaction coordinates and preparing a potential energy surface (PES). RC1 values are scanned from 3.55 to 1.35 and RC2 values are scanned from 2.00 to 0.80 using 0.1 increments for each coordinate. An independent two dimensional PES is prepar ed using RC3 and RC4 as in Chapter 3. The PES is constructed by scanning RC3 between values of 2.7 and 0.8 and RC4 between values of 1.2 and 5.0 using 0.1 increments for bo th coordinates. Using the most stable structure in the energetic valley of products of this RC3RC4 PES, a subsequent two dimensional scan is done using RC1 and RC2 as the reaction coordinate s. RC1 is scanned between values of 4.5 and 1.0 and RC2 is scanned between values of 2.0 and 0.8 4.3. Results and Discussion The PES shown in Figure 4-2 is prepared by scanning RC1 and RC2 and shows that Asp59 carboxylic acid transfers a proton to the glyc osidic oxygen atom of the ligand before the nucleophilic attack of Tyr342 to the anom eric carbon atom. Howeve r, the energy barrier for this reaction is very high (78.8 kcal/m ol) indicating that this reaction is not likely to happen. Also, the sudden change seen on the PES close to the covalent intermediate might indicate RC1 and RC2 are not proper reaction coordinates to monitor this reaction. One should still note that in the PES the covalent intermediate lies in an ener gy valley and is 11 kcal/ mol lower in energy than the Michaelis complex, indicating the stability of the covalent intermediate. The PES prepared scanning RC3 and RC4 inst ead, shown in Figure 4-3, gave a more reasonable picture in terms of en ergetics with a barrier of 40.2 kcal/ mol. However, this scan does not form a covalent intermediate directly at the end but a stable oxocarbenium ion (i.e. a

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83 Figure 4-1. Reaction coordinates us ed to generate poten tial energy surfaces. Th e sialic acid and lactose parts of sialyllactose are colo red in black and orange, respectively. Table 4-1. Properties of stationary points on potential energy surfaces Energy (kcal/ mol) RC1 () RC2 () RC3 () RC4 () Anomeric C to ring O distance () Pyramidalization around anomeric C () RC1-RC2 scan: Michaelis complex Transition state Covalent intermediate 10.9 89.8 0.0 3.45 1.85 1.45 1.70 0.81 0.95 1.42 1.52 3.79 2.59 2.58 0.98 1.40 1.40 1.40 -31.3 -16.9 29.0 RC3-RC4 scan: Michaelis complex Transition state Oxocarbenium ion Subsequent RC1-RC2 scan: Transition state Covalent intermediate 13.1 53.3 34.2 33.7 0.0 3.62 3.36 2.68 2.25 1.45 1.65 1.65 1.64 1.00 1.00 1.40 1.80 3.06 4.87 4.47 1.70 1.10 0.98 0.95 0.95 1.40 1.34 1.28 1.31 1.40 -32.5 -9.0 -0.4 7.8 28.8

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84 Figure 4-2. Potential energy surface for SN2-like mechanism of TcTS covalent intermediate formation. carbocation) is located on the PES. The oxocarbe nium ion formation is also confirmed by the planarity around the anomeric carbon atom a nd shortening of the bond between the anomeric carbon and its neighboring oxygen atom as seen in Table 4-1. The PES also shows that the hydrogen transfer from Asp59 carboxylic ac id to the oxygen of the methoxy group representing the glycosidic oxygenoccurs prior to the bond breaking between the methoxy group and the sialic acid. To complete the reaction, a subsequent two dimensional scan is performed using RC1 and RC2 as the reaction coordinates. The PES in Fi gure 4-4 shows that the oxocarbenium ion readily forms the covalent intermediate with a small energy barrier of 1.9 kcal/ mol. The covalent intermediate lies in an energy valley and is 3.2 kcal/ mol lower in energy than the Michaelis complex.

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85 Figure 4-3. Potential energy surface for SN1-like mechanism of TcTS that shows a stable oxocarbenium ion formation. A potential energy diagram for th e entire trans-sialidase reaction is constructed bringing all the information obtained from AMBER QM/MM calcu lations together and shown in Figure 4-5. We are able to reproduce the energetical plausibility of the covalent intermediate formation and confirm the oxocarbenium ion as a stable intermediateshown in Chapter 3 with ONIOM methodusing a different QM/MM method. 4.4. Conclusion The results of the two different QM/MM m et hodologies described in Chapters 3 and 4 agree on the energetic plausibility of the covalent intermediate formationshowing it is almost equienergetic with the Michaelis complexfo r both TcTS and TrSA. The trans-sialidase reaction of TcTS is found to proceed with an SN1-like mechanism. Here, the term SN1-like mechanism is used to describe that the cleav age of the bond between th e glycosidic O and the anomeric C occurs before the bond formation between the anomeric C and Tyr342. However, the Oxocarbenium ion

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86 Figure 4-4. Potential energy surface for SN1-like mechanism of TcTS that shows covalent intermediate formation from the oxocarbenium ion. Figure 4-5. Potential energy di agram of trans-sialidase react ion of TcTS using QM/MM. 40.2 18.6 18.6 40.2 -3.2 0.0 0.0 20.5 20.5 Sialic acid-OMe + TcTS Oxocarbenium ion + TcTS TcTS covalent intermediate Oxocarbenium ion + TcTS Reaction Coordinate Relative Energy (kcal / mol) + Methanol Sialic acid-OMe + TcTS

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87 mechanism is not a typical SN1 mechanism in which the cationic intermediate formation occurs further away from the nucleophile without any nucleophilic assistance. Instead, the nucleophile, Tyr342, stands in such a position in the active site to assist the oxocarbenium ion formation. As the reaction proceeds from the Michaelis comple x to the oxocarbenium ion, the anomeric C migrates away from the lactose and gets closer to Tyr342, which results in stabilization of the positive charge developed on the anomeric C by the polarized negative charge on the hydroxyl group of Tyr342. The spatial approach of Tyr 342 to the anomeric C, the increasing negative charge on Tyr342s hydroxyl O atom and the decreasing positive charge on the anomeric C during this process (shown in Ta ble 3-1) support this view altoge ther. Overall, the active site modifies an otherwise SN1 mechanism with nucleophilic participation and the new mechanism is a complex mechanism which has both SN1-like and SN2-like properties. This reaction is an example of the large spectrum of reaction mechanisms that lie between a strict SN1 mechanism and a strict SN2 mechanism. 128,129 Both of our methodologies point to a stable oxocarbenium ion intermediate, although with a small energetic barrier for collapsing into a co valent intermediate. Our calculations also show that the rate determining step in the TcTS mechanism is the cleavage of the bond between the lactose and the sialic acid. For the rate determini ng step, a late transition structure similar to an oxocarbenium ion is anticipated by the potential energy diagrams obtained. These results agree with the KIE results that were interpreted to be indicative of a transition structure with significant nucleophilic participation and little charge development.

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88 CHAPTER 5 MOLECULAR DYNAMICS STUDY OF TRYPANOSOMA cruzi TRANS-SIALIDASE AND TRYPANOSOMA rangeli SIALIDASE 5.1. Introduction Enzym es are far from being static structures and their dynamics can play significant roles in catalysis. The range of dynamics can extend from small conformationa l changes of a single side chain to correlated motion of one or more regions of the enzyme130-135 with time scales in the nanosecond to millisecond region and can be re sponsible for activatio n/inactivation of an enzyme. Difference in catalytic functiona lity despite the high structural similarity point to minute differences between Trypanosoma cruzi trans-sialidase (TcTS) and Trypanosoma rangeli sialidase (TrSA) which may be related to differen ces in the dynamics of the two enzymes. Thus, the dynamical properties of the two active sites will be thoroughly i nvestigated in this chapter by monitoring measurable properties fo r the all-atom simulated systems of free and ligated forms of TcTS and TrSA. There are a number of questions which we focus on. The first and main question is what structural or dynamical differe nces between the two enzymes can cause the difference in catalytic activities. The second one arises from the recent study that succeeded to transform TrSA which has no trans-sialidase activity into a form that shows 10% of the trans-sialidase activity of TcTS using onl y 5 point mutations (TrSA5mut: Met95Val, Ala97Pro, Ser119Tyr, Gly248Tyr, Gln283Pro).72 Since none of these five residues are directly im plicated in the chemical catalysis step, the change of confor mational dynamics and/or structure due to these mutations should be responsible for the acquired trans-sialidase activity. Using the available Xray crystal structures of all three enzymes wild type TcTS, wild type TrSA and TrSA5mutas

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89 starting structures, a comparativ e study of all-atom molecular dynamics (MD) simulations of these systems can shed some light into their differences in detail. The third question arises from the differe nce in DANA binding properties of TcTS and TrSA. DANA, a common inhibitor for sialidases is significantly less efficient for TcTS (Ki= 12.29 mM) compared to TrSA (Ki= 1.5 M).72 Detailed analysis of MD simulations of DANAbound TcTS and TrSA can shed some light on the reasons of this difference. The fourth question is related with the experi mental results showing that lactose which acts as a sialic acid acceptor can not bind TcTS in the absence of sialic acid in the medium.75 This observation brings to mind the possibility of a stru ctural change or a change in the dynamics in the active site due to occupation of the sialic acid binding site which will favor subsequent lactose binding. A thorough comparison of free TcTS and TcTS covalent intermediate MD simulations will be made searching for any structural or dynamical changes due to covalent intermediate formation or DANA binding that ca n modify the active site to a more proper binding site for lactose. We also investigate how TrSA binds sialyllact ose since there is no X -ray crystal structure available. The active site residues are listed for TcTS a nd TrSA in Figure 5-1. Only 12 mutations are found out of 43 residues in the activ e sitewhen defined as the resi dues that have at least one atom in 10 of Tyr342s hydroxyl O atom. From now on, the residue numbering of TcTS will be used for corresponding residues of both enzymesunless otherwise statedto prevent confusion and to provide better means to compare the two enzymatic systems.

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90 TcTS Arg35 Leu36 Pro37 Asp51 Arg53 Phe58 Asp59 Asn60 Val95 Asp96 TrSA Arg39 Ile40 Pro41 Asp55 Arg57 Phe62 Asp63 Asn64 Met99 Asp100 TcTS Pro97 Tyr119 Trp120 Leu176 Gly177 Gly178 Ala179 Gly180 Gln195 Ser229 TrSA Ala101 Ser123 Trp124 Val180 Gly181 Gly182 Val183 Gly184 Gln199 Ser233 TcTS Glu230 Pro231 Val232 Arg245 Asp247 Tyr248 Arg251 Pro283 Gly284 Ser285 TrSA Glu234 Pro235 Ala236 Arg249 Asp251 Gly252 Arg255 Gln287 Asp288 Ser289 TcTS Gln286 Ser287 His304 Pro305 Trp312 Arg314 Ser340 Ala341 Tyr342 Ser343 TrSA Gln290 Ser291 His308 Pro309 Trp316 Arg318 Ser344 Gly345 Tyr346 Ser347 TcTS Glu357 Glu362 Tyr364 TrSA Glu361 Asp366 Tyr368 Figure 5-1. Corresponding active site residues of TcTS and TrSA that have at least one atom within 10 radius of Tyr342 hydroxyl O atom. The sequences are aligned using Basic Local Alignment Search Tool (BLAST).136 The residues that are different between the two enzymes are underlined a nd original numbering in each enzyme is used. 5.2. Methods 5.2.1. System Setup and Simulation Details The initial structur es of TcTS and TrSA are obtained from the Protein Databank ( www.rcsb.org PdbID: 1MS3, 2AH2, 1MS1, 1S0I, 1N 1S, 2A75, 1N1T, 1W CS). All these 8 structures correspond to different points in the reaction coordinate (Table 5-2). Only the catalytic domainthe first 371 residues for TcTS, the first 375 residues for free and DANA-bound TrSA, the first 373 residues for TrSA c ovalent intermediateand the liga nd, if any, are kept and the rest of the proteins and any solvent molecules are excluded wh ile preparing the systems for simulation (Figure 5-2). In additi on to the above 8 structures avai lable in the Protein Databank, the missing sialyllactose-bound form of TrSA is modelled in silico superimposing DANA-bound TrSA (1N1T.pdb) and the sialyllactose-bound TcTS in VMD program137 and imitating the sialyllactose pose seen in TcTS. While preparing the input files for MD simula tions several changes have been performed on the original X-ray crystal structure files to im itate the natural catalysis event better. In the systems that have the Asp59Ala mutation, the alan ine59 is mutated back to an aspartic acid to

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91 obtain the wild type enzyme. If the X-ray crys tal structure is obtaine d as a homodimer, only chain A is preserved and chain B is deleted. And for the covalent intermediates, the F atom inserted into sialic acid to be able to obtain a stable crystal of the covalent intermediate is changed to a H atom as in the natural catalysis event. The resulting pdb file is fed in to the LEAP module of AMBER9117 molecular dynamics package where the necessary H atoms and any other missing atoms are added as well as being solvated with 23,000 TIP3P118 explicit water molecules and truncated octahedral boundary conditions. Since the Asp59 is responsible for acid/base cat alysis, its protonation state is changed to the aspartic acid form instead of the aspartate form which is found normally under physiological conditions. The protonation states of Asp59, Glu230 and Tyr342 change over the course of the reaction and are adjusted in the covalent interm ediates accordingly (Table 5-1): Asp59 is in the aspartate form while Glu230 is in the glutamic acid form instead of the glutamate form due to the H transfer from Tyr342 to Glu230 in the reacti on. In all other systemsfree enzymes and Michaelis complexesAsp59 and Tyr342 are pr otonated while Glu230 is unprotonated. Amber.FF99SB120,138 and Glycam04121-123 force fields are used to construct the topology files. Since the inhibitor DANA is not a standard residue in Glycam04, the parameters for this molecule had to be constructed. First the geom etry for DANA is obtained modifying the standard terminal sialic acid geometry from Gly cam04 using HYPERCHEM program and minimizing it to get planarity around its anomeric C atom. HF/6-31G* in GAUSSIAN0399 package is used to produce RESP charges for DANA and these charges are then fed into the ANTECHAMBER module of AMBER9 package to produce th e parameter and topology files using GAFF139 force field.

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92 The PMEMD module of AMBER9 package is used first to relax the systems under periodic boundary conditions with a procedur e explained in detail as follows: (1) 1000 steps of minimization using steepest descent method using a cutoff of 8.0 for truncation of nonbonded pa irs while restrain ing all backbone atoms of the enzyme with a restraint of the form k ( x)2 where the force constant is 5.0 kcal/mol 2. (2) 15 picoseconds of molecu lar dynamics with a timestep of 1 femtosecond at 300 K and constant pressure using periodic boundary conditions and Berendsen weakcoupling algorithm126 with a coupling time constant of 0.5 picoseconds and a pressure relaxation time of 0.5 picosec onds to provide constant temperature. SHAKE algorithm is also used to cons train bonds involving hydrogen atoms. The constraints for the backbone atoms and the nonbonded cutoff value used in the 1st step are kept. (3) Three subsequent minimizations with th e same procedure as in step 1 except decreasing the force constant for th e backbone atoms to 2.0 kcal/mol 2, 0.1 kcal/mol 2 and finally to 0, respectively. (4) 5 picoseconds of molecular dynamics with the same procedure as in step 2 except decreasing the restraint force constant to 1.0 kcal/ mol 2. (5) 15 picoseconds of molecu lar dynamics with the same procedure as in step 2 except decreasing the restraint force constant to 0.5 kcal/ mol 2. (6) 1 nanosecond of molecular dynamics with the same procedure as in step 2 except releasing the restraints and increasing the timestep to 2 femtoseconds. Following the relaxation, 50 ns of production run is performed following the same procedure as in the last step of the relaxation. 5.2.2. Analysis Methods The residu e numbering for the analysis is adjusted for all systems simulated to prevent any confusion and to provide consistency between different systems as described in Figure 5-2. The residue numbering in TcTS is used for all syst ems throughout this chapter and Chapter 6 except otherwise stated. While comparing the average structures of MD simulations, all residues that have at least one atom in 10 of Tyr342s hydroxyl O atom are included. The conformations of the residues in the protein are monitored through 1 and 2 dihedral angles which are defined in Figure 5-3. The histograms of 12 populations of Trp312

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93 and Tyr119 are prepared using 10 X 10 grids. The histograms of 12 populations of Tyr342 and Leu36 are prepared using 5 X 5 grids. Table 5-1. Properties of simulated structures PdbID 1MS3 2AH2 1MS1 1S0I 1N1S 2A75 1N1T 1WCS Definition TcTS TcTS cov.int. TcTSDANA TcTSSLT TrSA TrSA cov.int. TrSADANA TrSASLT TrSA5mut Resolution () 1.65 1.60 1.80 1.60 1.64 1.95 1.60 2.80 Extra molecules in active site in X-ray crystal structure Glycerol Glycerol SO4 Mutations to get X-ray crystal structure N58F N58F N58F N58F, D59A Double conformation s in X-ray structure in the catalytic domain L36, S115, S116, S118, Y119, S122, H123, S257, Y342 E9, K12, I49, F58, D59, Y119, M170, T262, L264, M297 L36, D96, Y119, Y342 K11, F58, N60, E167 Prot. State of Asp59 in MD + + + + + + + Prot. State of Tyr342 in MD + + + + + + + Prot. State of Glu230 in MD + + The extra molecules found in the active site in X-ray crystal structures whic h are part of the solvent are also designated. The + and represent aspartic acid and aspartate forms of Asp59 (and likewise for other residues), respectively. Only the mutations in the catalytic domain are mentioned in this table. The salt bridges which reside in 10 radi us of Tyr342s hydroxyl oxygen are determined with the Saltbridge option of VMD. In this option, a salt bridge is considered to be formed if the distance between the oxygen atoms of acidic residue s (aspartic acid and glutamic acid) and the nitrogen atoms of basic residues (arginine, histidine and lysine) are within the cutoff distance

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94 3.2 used herein at least one frame. The resu lts are used as a starting point to monitor the important hydrogen bonding interactions around th e active site and additionally glutamine, asparagine and tyrosine residues closeby are included in the hydrogenbond analysis. Hydrogen bonds among the residues are monitored with ptra j module of AMBER9. The criteria used for hydrogen-bonding are that heavy atom to heavy atom distance is 3.5 or less and 1st heavy atom-hydrogen-2nd heavy atom angle is 140 or larger. Figure 5-2. The regions of the Tc TS and TrSA that are used for MD simulation and analysis. The red regions are the lectin-like domains ex cluded in the MD simulations. The blue regions are the amino acid inserts that di ffer between TcTS and TrSA and so are excluded in residue numbering in the MD analysis part to provide consistency between the two enzymes and prevent confusion. Figure 5-3. Description of 1 and 2 dihedral angles of a residue in a protein. is the dihedral angle of Cprecedent residue-N-C-C, is the dihedral angle of N-C-C Nsubsequent residue, 1 is the dihedral angle of N-C-C-C, 2 is the dihedral angle of C-C-C-C 1. The dihedral angles being 0 means that the terminal atoms are oriented cis to each other. 1 371 631 375 634 24 21 4 1 1 372 631 1MS3, 2AH2, 1MS1, 1S0I 1N1S, 1N1T, 1WCS 2A75

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95 Ni i iN RMSD1 21 (5-1) tt t jj jxtx t RMSF0 2))(( 1 (5-2) The root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values are also calculated with ptraj module in AMBE R9. RMSD is a custom method to measure the overall similarity of 3-dimensional structur es. RMSD is defined with Eq. 5-1 in which i is the distance between a pair of equivalent atoms in the two structures being compared and N is the number of particles included in the calculation. The minima l RMSD value is found trying different superpositions. In our study, all C atoms of the enzyme are included in this calculation giving a measurement of the deviation of the en zyme over time from the starting structure. On the other hand, RMSF is a measurement of deviat ion of a particle from a reference structure average structure of production run in our caseover time. RMSF is defined with Eq. 5-2 where t is time and x is the time-averaged position of the same particle. RMSF can be used as a measure of the mobility of a particle in a simulati on and is related to experimental B-factors. One can relate RMSF from simulations to experime ntal B-factors by the relationship given in Equation 5-3. B-factor=8 2 (RMSF)2 (5-3) Ptraj module of AMBER9 and VMD are both us ed to monitor the distances, angles and dihedral angles in the simulations. 5.3. Analysis 5.3.1. Stability of the Mol ecular Dy namics Simulations The root mean square deviation (RMSD) w ith respect to the starting geometries is generally used to show the stability of the simu lations. Figure 5-4 show s that the RMSD value of C atoms of the protein with respect to the st arting structure of the production MD simulation

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96 does not exceed 2.5 in any simulation as well as indicating that the systems in our simulations are stable. 5.3.2. Root Mean Square Fluctuation (RMSF) Analysis of MD Simulations of TcTS and TrSA In this sec tion, the root mean square fluctuations (RMSF) of C atoms of the enzymatic systems will be thoroughly investigated. Since the loops extending from residue 142 to residue 147 and from residue 123 to 128 are more mobile for all TrSA species compared to all TcTS species, these loops will not be mentioned in each comparison repeatedly. Also, the mobilities of the loops extending in the ra nge 22-25, 142-147, 163-169, 257-263 and 295-296, which are all far from the active site, will not be paid much a ttention due to their distance and irrelevance to the active site although their mobili ties vary in each simulation. 5.3.2.1. Comparison of RMSF changes due to covalent intermediate formation in TcTS and TrSA The main difference between TcTS and TrSA in RMSF changes due to covalent intermediate formation is seen for the RMSF of the loop bearing Pro283 in TcTSwhich is equivalent to Gln283 in TrSA (Figure 5-1). Comp aring Figures 5-5 and 5-6 shows that there is significant RMSF increase for this loop in TcTS due to the covalent intermediate formation while a significant RMSF decrease occurs in the case of TrSA. A similar trend is observed for the loop bearing Tyr248 in TcTS which is equivalent to Gl y248 in TrSA. The loop bearing Tyr248/Gly248 lies right next to the loop bearing Pro283/Gln283. Another difference is observed for the loop bearing Trp312. In TcTS, lower RMSF values of this loop are seen in the covalent intermediate compared to the free enzyme, while the reverse trend is seen in TrSA. This di fference can be related to the fact that the loop bearing Trp312 moves outwards into the exterior region formi ng a more open active site in TrSA covalent intermediate and unligated TcTS MD simulations, while this loop remains mainly intact in TcTS

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97 Figure 5-4. Root mean square de viation (RMSD) of MD simulations with respect to the initial structure of the production runs. A) TcTS species B) TrSA species

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98 covalent intermediate and unligated TrSA MD simulations. This active site opening will be discussed later in this chapter. For the -strand bearing Asp96, TrSA covalent intermediate has higher RMSF values compared to free TrSA, wh ile almost no difference is seen between free TcTS and TcTS covalent intermediate. 5.3.2.2. Comparison of RMSF between DANA-bound TcTS and Tr SA simulations When the RMSF values of DANA-bound forms of TcTS and TrSA catalytic regions are compared, the only difference is seen in th e three loops bearing Tyr248, Trp312 and Asp96, being higher in DANA-bound TrSA (Figure 5-7 and 5-8). For both TcTS and TrSA, DANA binding promot es a decrease in the mobilities of the three loops bearing Tyr248, Pro283 and Trp312 (Figure 5-7 and 5-8). 5.3.2.3. Comparison of RMSF between wild-type TcTS, wild-type TrSA and TrSA5mut simulations The RMSF values of TrSA5mut for the three loops bearing Tyr248, Pro283 and Tyr119 are lower compared to wild type TrSA, and are cl ose to the values observed for wild-type TcTS (Figure 5-9). TrSA5mut has lower RMSF values for the loop bearing Trp312 compared to TrSA, too, however, corresponding RMSF values for wild -type TcTS are much higher than both TrSA forms. The locations of three out of the five point mutations (Met95V al, Ala97Pro, Ser119Tyr, Gly248Tyr, Gln283Pro) performed on TrSA are obser ved to change their local mobility and approach to that of TcTS. No effect on the lo cal flexibility of the two mutations surrounding Asp96 is observed. 5.3.2.4. Comparison of RMSF between sial yllactose-bound TcTS and sialyllactose-bound TrSA There are two main differences between the RMSF values of the active sites of sialyllactose-bound forms of TcTS and TrSA (F igure 5-10). The loop be aring Pro283 has higher RMSF in sialyllactose-bound TcTS than sial yllactose-bound TrSA, wh ile the loop bearing

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99 Trp312 has higher RMSF in sialyl lactosebound TrSA as seen in Figure 5-10. The high RMSF values for the loop bearing Trp312 is related to reorientation of Tr p312 to adopt the binding conformation and will be explained in detail in the discussion of sialyllactose binding of TrSA. 5.3.2.5. Comparison of experimental B-factor s of free and covalent intermediate forms of TcTS and TrSA The com parison in Figure 5-11 shows that Bfactors of free TcTS and TcTS covalent intermediate are almost identical except from that the loop bearing Asp59 is more flexible in the covalent intermediate. The comparison of Bfactors of free TrSA and TrSA covalent intermediate (Figure 5-12) does not really inform us about the flex ibility changes due to covalent intermediate formation since the resolution of them are different.64 and 1.95 respectively. 5.3.2.6. Comparison of experimental B-fa ctors between DANA-bou nd TcTS and TrSA Xray crystal structures No noticeable effect of DANA binding is seen on the B-factors of the active site in TcTS (Figure 5-13). The same trend is seen in Figure 5-16 for TrSA. The comparison of Figure 5-13 and Fi gure 5-16 shows that DANA-bound TcTS has higher B-factors than DANA-bound TrSA for the three loops bearing Tyr248, Pro283 and Trp312. Additionally, the loop bearing Asp59 ha s higher B-factors in DANA-bound TrSA than in DANA-bound TcTS. 5.3.2.7. Comparison of experimental B-fact or s between wild-type TcTS, wild-type TrSA and TrSA5mut X-ray crystal structures The trace of B-factors of TrSA5mut is very flat compared to TcTS and TrSA due to its lower resolution (Figure 5-15). Since the resolution of wild-type TcTS, wild-type TrSA and TrSA5mut are 1.65 1.64 and 1.95 respectively, we can only compare the B-factors of TcTS and TrSA. The three loops bearing Tyr248, Pro283 and Trp312 have higher RMSF values in TcTS

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100 Figure 5-5. C RMSF comparison of free TcTS with TcTS covalent intermediate. The arrows show the RMSF differences in the active site. Figure 5-6. C RMSF comparison of free TrSA with TrSA covalent intermediate. The arrows show the RMSF differences in the active site. Trp312 Gln283 Gly248 Asp96 Tyr248 Pro283 Trp312 Tyr248 Pro283 Trp312 Tyr248

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101 Figure 5-7. C RMSF comparison of free and DANA-bound forms of TcTS. The arrows show the RMSF differences in the active site. Figure 5-8. C RMSF comparison of free and DANA-bound forms of TrSA. The arrows show the RMSF differences in the active site. Trp312 Pro283 Tyr248 Gly248 Gln283 Trp312

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102 Figure 5-9. C RMSF comparison of TrSA, TcTS and TrSA5mut. The differences in the active site are indicated with arrows. Figure 5-10. C RMSF comparison of sialyllactosebound forms of TcTS and TrSA. The differences in the active site are indicated with arrows. Tyr/Gly248 Pro/Gln283 Trp312 Tyr/Ser119 Pro/Gln283 Trp312

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103 while the loop bearing Tyr119 has higher RMSF values for TrSA. 5.3.2.8. Comparison of RMSF values from MD simulations and B-factors from X-ray crystal stru ctures The analysis of RMSF values of MD simulations showed higher mobility in TrSA than TcTS for the loops bearing Tyr248, Pro283 a nd Trp312, while the experimental B-factors showed higher mobility for these loops in TcTS. Th ese 3 loops reside back to back at the outer surface of the enzymes. The reason of this inco nsistency is most likely the crystal contacts observed for these three loops in TrSA. When th e crystal contacts are in vestigated using the symmetry operations on X-ray crysta l structure with SwissPdbViewer,140 strong interactions from a neighboring enzyme is observed for a ll these 3 loops constraining their motion and resulting in unnatural and low B-f actors for TrSA (Figure 5-14). No such contacts exist for TcTS X-ray crystal structur e in these three loops. Thus, the RMSF results obtained from MD simulationswhich are free from the crystal contact problemshould be more reliable for these regions. Based on the RMSF analysis results, we observe that these three l oops are more rigid in TcTS compared to TrSA due to the nonide ntical residues, namely Tyr248/Gly248 and Pro283/Gln283, in these particular loops. This rigidity is important since Trp312 forms the aglycon binding site of the catalytic cleft. The two loops b earing Tyr248 and Pro283 are found to stay intact even in free TcTS MD simulation, in which active site opening due to outwards motion of Trp312 loop is observed. 5.3.3. Comparison of Average Structures fr om MD Simulations of TcTS in Ligated and Unligated Forms In this sec tion, the average structures of 50ns MD simulations of different forms of TcTS are compared. Overall, they look very similar with RMSD values less than 1.1 when their backbone atoms are superimposed. Only the residue s that adopt different conformations in the

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104 Figure 5-11. Comparison of crystal structure C B-factors of TcTS covalent intermediate with its free form. The difference in active site B-factors is shown with an arrow. two structures being compared are mentioned and a ll others can be assumed to be very similar. 5.3.3.1. Comparison of unligated TcTS with TcTS covalent intermediate average structures Tyr342, Glu230, Asp59, Leu36 and Gln286 confor m ations differ due to change of 1 dihedral angles. Ser287 conformation is also different due to a slight backbone motion. Trp312 is found in the binding conformation in the covalent in termediate while the loop opening observed in unligated TcTS results in a more open active site and different positioning of Trp312. In both structures Tyr119 is in the down conformation (Figure 1-9). It shoul d also be noted that covalent intermediate formation induced a sli ght outwards motion in the loop structure causing Tyr119 backbone to lie highe r in the active site. 5.3.3.2. Comparison of unligated TcTS with DANA-bou nd TcTS average structures Tyr119 and Leu36 conformations differ in unligated and DANA-bound forms of TcTS due Asp59

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105 Figure 5-12. Comparison of crystal structure C B-factors of TrSA covalent intermediate with its free form. Figure 5-13. C B-factor comparison of free and DAN A-bound forms of TcTS X-ray crystal structures. The arrows indicate the lo ops whose mobility differ between DANAbound forms of TcTS and TrSA. Tyr248 Trp312 Asp59 Pro283

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106 Figure 5-14. C B-factor comparison of free and DAN A-bound forms of TrSA X-ray crystal structures. Figure 5-15. C B-factor comparison of TcTS, TrSA and TrSA5mut X-ray crystal structures. Gly248 Gln283 Trp312 Asp59 Tyr/Ser119 Tyr/Gly248 Pro/Gln283 Trp312

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107 Figure 5-16. Crystal contacts of TrSA ar ound the loops bearing Trp312, Gln283 and Gly248 (shown in licorice form). Tyr342 is also shown in licorice form to clarify relative orientation of the active site. A neighbor ing enzyme (its backbone shown in red) limits the mobility of these three loops. to changing 1 dihedral angles. Tyr119 is in the up conformation (Figure 1-9) in DANA-bound TcTS and DANA-binding induces a small change in the loop structure of Tyr119 causing its backbone to lie higher in the active site. Tyr 342 has moved up to interact with DANA in the DANA-bound enzyme, which also causes the loop bearing Tyr342 to move up and the Ala341 conformation to change. Trp312 is found in the binding conformation in the DANA-bound TcTS. Overall, we observe that DANA-binding locks the arginine triad, Tyr342, Glu230 and Trp312 into position to embrace the ligand tightly. 5.3.3.3. Comparison of unligated TcTS with sialyllactose-bound TcTS average structures When the average structures of unligated and sialyllactose-bound for ms of TcTS are compared, Glu230, Tyr119, Asp59, Leu36 and Gl n286 conformations differ due to changing 1 dihedral angles. Ser287 and Ser288 conformations are also different due to a slight loop motion. In the sialyllactose-bound TcTS, Tyr119 is in th e up conformation (Fi gure 1-9) and the loop bearing Tyr119 approaches a little to the ligand. Tyr342 1 is different in the complex to interact with the ligand, the loop b earing Tyr342 changes its conformation slightly and Ala341

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108 conformation is different. Glu230 has moved to a conformation unfavorable for a hydrogenbonding interaction with Tyr342. Asp59 position is also different in the complex due to loop motion on top of conformational change. Trp312 is found in the bindi ng conformation in the complex. Overall, sialyllactose binding also lo cks the arginine triad, Tyr342, Trp312 and Tyr119 into position to embrace the ligand tightly. Howe ver, Asp59 is not found in a conformation to interact with O3 atom of the galactose part of the ligand (glycosidic O in Figure 1-8) and Glu230 is not hydrogen-bonded to Tyr342. 5.3.4. Comparison of Average Structures fr om MD Simulations of TrSA in Ligated and Unligated Forms In this sec tion, the average structures of 50ns MD simulations of different forms of TrSA are compared. Overall, they look very similar with RMSD values less than X when their backbone atoms are superimposed. Only the residue s that adopt different conformations in the two structures being compared are mentioned and a ll others can be assumed to be very similar. 5.3.4.1. Comparison of unligated TrSA with TrSA covalent intermediate average structures Ile36, Gln283 and Tyr342 confor mations di ffer between the unligated TrSA and the covalent intermediate due to changing 2 dihedral angle. Asp59 conformation is also different due to 1 difference. Trp312 is in the binding conformation in unligated TrSA while its conformation has changed on top of a slight out wards movement of its loop in the covalent intermediate. The loop bear ing Tyr342 stays intact even after covalent intermediate formation. The arginine triad rearranges in the covalent intermediate to em brace the carboxylate of the sialic acid tightly. Also, the loop bearing Asp247 appr oaches more to the sialic acid and Asp247 conformation changes to interact with Arg245 in the covalent intermediate.

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109 5.3.4.2. Comparison of unligated TrSA w ith DANA-bound TrSA average structures Ile36 and Gln283 conformations differ betw een the unligated TrSA and the DANA-bound TrSA due to changing 2 dihedral angle. Asp59 conforma tion is also different due to 1 difference. The arginine triad rearranges in the DANA-bound TrSA to embrace the carboxylate of the sialic acid tightly. Also, Trp312 conforma tion resembles more to the binding conformation with only a slight change in its loop, and the loop bearing Asp247 lies closer to the sialic acid in DANA-bound TrSA. 5.3.4.3. Comparison of unligated TrSA with sialyllactose-bound TrSA average structures Ile36 and Gln283 conform ations differ between the unligated TrSA and the sialyllactosebound TrSA due to changing 2 dihedral angle. Phe58 and Asp59 conformations are also different due to 1 difference. The Phe58 conformation in sialyllactose-bound TrSA provides Phe58 to embrace the lactose part of the ligand. The arginine triad rearranges in sialyllactosebound TrSA to embrace the carboxylate of the sia lic acid tightly. Sialyllactose binding also promotes Trp312 conformation to change to the binding conformation and movement of its loop to make stacking interactions with the lactose part of the ligand. Th e loop bearing Asp247 and the loop bearing Ser119 are also closer to the active site in sialyllactose-bound form. 5.3.5. Comparison of Sialyllactose-Bound Tc TS and Sialyllactose-bound TrSA Average Structures The sialyllactose pose is very similar in th e two enzymes. In the active sites, the conformations of Phe58 and Asp59 differ for the tw o enzymes. There is also a slight difference in the loop bearing Tyr342 which might be related to presence of Ala341 in TcTS instead of Gly341 in TrSA. There are also several mutatio ns that are constrai ning the active sites differently: (i) Val95 in TcTS is Met95 in TrSA which resu lts in a more restricted active site in TrSA around O4 atom of sialic acid.

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110 (ii) Leu176 in TcTS is Val176 in TrSA which results in a more restricted active site in TcTS around N-acetyl group of sialic acid. (iii) Pro283 in TcTS is Gln283 in TrSA which can affect the Trp312 conformation. 5.3.6. Comparison of DANA-bound TcTS w ith DANA-bound TrSA Average Structures The main difference between the two average st ructures is that the glycerol branch of DANA ( 3 in Figure 1-6) adopts di fferent conformations in the two enzymes. Asp59 conformation is also different due to 1 change. Asp59 conformation differs due to 1 change. There is also a slight difference in the loop bearing Tyr342 which might be related to the presence of Ala341 in TcTS instead of Gl y341 in TrSA. Trp312 is seen in the binding conformation in DANA-bound TcTS. 5.3.7. Behavior of Key Residue s of TcTS and TrSA in MD Am ong the key residues in the active site, Trp312 and Asp59 are on flexible loops, Tyr119 resides on a very short 3-10 helix which is on a flexible loop, Glu230 and Asp96 lie on -strands which are parts of the -propeller architecture that is char acteristic for neuraminidases while Tyr342 lies on a small loop surrounded by -strands. The conformati ons and overall motion of these key residues will be investigated in this section. 5.3.7.1. Behavior of Trp312 The conform ation of Trp312 is found very diffe rent in TcTS and TrSA X-ray crystal structures which can be distinguished by both 1 and 2 dihedral angles. Th e behavior of Trp312 side chain is monitored preparing a 2-dimensional histogram of its 1 and 2 dihedral angles during the simulations. The histograms in Figur e 5-17 show that there is a dominant binding mode of Trp312 which can be described around the region of 1= -50 and 2= -75. Among TcTS species, the binding mode is highly populated by Trp312 in DANA or sialyllactose-bound cases while in the free enzyme or the covalent intermediate the binding mode is visited much less although it is still the dominant conformation.

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111 Interestingly, Trp312 also visits the binding mode in the free and sialyllactose-bound TrSA although populating it to a lower ex tent compared to the TcTS simulations (Figure 5-18). The reasons of this population diffe rence are found to be related to the starting conformation ( 1= 50 and 2= 75) obtained from the X-ray cr ystal structures as well as the flexibility difference in this region between TcTS and TrSA and will be discussed later in this chapter. In MD simulations of the covalent intermediate and the DANAbound forms of TrSA as well as the TrSA5mut, Trp312 is seen to sample different conf ormations which are different in 1 or 2 dihedral angle from the binding mode seen in TcTS but closer to their initial conformation. Since Trp312 resides on a flexible loop, the conformation and the motion of the backbone is also monitored through the RMSD of the backbone atom s of the loopresidues 307 to 317 (Figure 519). The high RMSD values for free TcTS and Tr SA covalent intermediate indicate significant change in the architecture of the loop. A similar motion is observed for TcTS covalent intermediate and TcTS-DANA although to a much smaller extent. The visualization of the allatom simulation shows that the high RMSD values are an indicator of a loop motion which forms a more open active site. However, when all the av ailable X-ray crystal st ructures of empty and ligated TcTS and TrSA are superimposed, the backbone of the loop bearing Trp312 is seen to adopt the same pose in all. A closer look shows th at both ends of the loop are attached at Lys309 and Arg314 strongly to neighboring residues with two strong H-bonding interactions. Especially, the salt bridges strongly connecting several loops together to finally attach to Lys309 is interesting. Lys309 side chain amino group extends to form H-bonds to both Asp337 and Asn339, while Glu338 in between forms a salt bridge to Arg7. This chain of strongly jointed residues can induce motion of the loop or its i mmobilization under differe nt circumstances. Since

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112 Arg7 lies close to the N-terminal of TcTS, the position of N-terminal is important for the loop bearing Trp312. Figure 5-17. Histograms of Trp312 12 populations in MD simulations for TcTS species. Dihedral angles are in degrees. The mobility of the loop bearing Trp312 seen in MD simulations, which is reflected neither in multiple conformations nor in larger B-factors for the loop in the X-ray crystal structure, is of curiosity. The reason of this difference can be re lated to the close crystal contacts observed. For example, in the X-ray crystal stru cture of empty TcTS, the two monomers forming

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113 Figure 5-18. Histograms of Trp312 12 populations in MD simulations for TrSA species. Dihedral angles are in degrees. the homodimer lie very close to each other around th is N-terminal region as well as some other regions. Direct H-bond distances le ss than 3.5 and water mediat ed H-bond distances less than

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114 3 are observed at the contact surface around the N-terminal region and around residues 336 and 338. These close contacts can serve to lock the N-terminal to a certain position which will also implicate a certain position for the loop of Trp312 in the X-ray crystal structures. Another point to mention is that the openi ng of Trp312 loop is only seen after the Tyr119 1 angle takes the value of ~180 (the down conformation of Ty r119, Figure 1-9). (Figure 5-20) It becomes easier for Trp312 and its loop first to move backwards and then move randomly forming a more open active site only after the st acking interaction of Trp 312 with Tyr119 is lost due to Tyr119 side chain swi nging down into the active site. 5.3.7.2. Behavior of Tyr119 Dual conform ation is observed for Tyr119 residue whic h lies at the periphery of the active site in X-ray crystal structures of free, DANA-bound and covalent intermediate forms of TcTS. The two conformations observed differ in 1 dihedral angle; in one c onformation Tyr119 side chain swings down into the active site while in the ot her conformation it points up extending into the exterior medium (Figure 1-9). Figure 5-21 shows the histograms of Tyr119 12 dihedral angles in MD simulations of TcTS species and TrSA5mut. Different 2 angles at the same 1 dihedral angle represent a flipping of the phenyl ring aro und itself. The histograms show a very localized down conformation for free and covalent intermediate forms of TcTS and TrSA5mut and a very localized up conformation for the sialy llactose-bound TcTS. Interestingly, in DANA-bound TcTS simulation, Tyr119 visits 3 di fferent conformations which differ in 1. The third conformation also extends out into the exterior medium. Another point to notice is that the down conformation 1= 180 must be very tightly held due to its interactions with the enzyme that even phenyl-ring flipping (2 change) is not observed. Since Tyr119 resides on a very short 3-10 helix which is surrounded by flexible loops, the conformation and motion of the loop is also monitored through the RMSD of the backbone

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115 Figure 5-19. RMSD (in ) of the loop that bear s Trp312 in MD simulations without fitting to the initial frame. Simulation time (ns)

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116 Figure 5-20. Correlation of RMSD of the backbone of Trp312s loop w ithout fitting to the initial conformation and the 1 dihedral angle of Tyr119 dur ing MD simulations. RMSD values more than 3 implicating a loop opening, is only observed in case of Tyr119 conformation in which the hydroxyl group swings down into the active site ( 1= ~180). atomsresidues 114 to 124 (Figure 5-22). The hi gher RMSD values observed for covalent intermediate and DANA-bound forms of TrSA as well as the TrSA5mut for the loop of

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117 Ser119/Tyr119 are indicators of a change of loop architecture a lthough not significant as confirmed by visualization of the MD simulations No such motion is observed for any of TcTS MD simulations. 5.3.7.3. Behavior of Phe58 Since different Phe58 conformations ar e observed for sialyllactose-bound TcTS and TrSA average structures of the simulations and du al conformations are obse rved in X-ray crystal structures of both TcTS and TrSA covalent intermediates, the 1 change of Phe58 is also monitored. In fact, this phenylalanine which exists in both TcTS and TrSA X-ray crystal structures is not a natural re sidue for TcTS, which indeed has an asparagine residue instead75. This Phe58 is one of the 7 mutations performed to obtain stable diffraction-quality crystals. All the other 6 mutations are in the lectin-like part which is not included in our simulations. The 1 dihedral angles in Figure 5-23 show that Phe 58 visits all three possibl e conformations in all simulations except two. In sialyllactose-bound Tr SA simulation, Phe58 adopts one conformation ( 1= 180), different from the initial one, and retain s it for the rest of the simulation. And in sialyllactose-bound TcTS simulation, Phe58 very ra rely samples the conformation that allows the stacking interaction and strongly prefers the other two possibl e conformations (Figure 5-23). Visualization of the trajectory file reveals that the conformation of Phe 58 seen in sialyllactosebound TrSA provides a stacking inter action with the lactose part of the sialyllactose. Phe58 conformation will be discussed in more detail later in this chapter. 5.3.7.4. Behavior of Asp59 1 dihedral angle change af fects whether Asp59 carboxylic acid group swings towards glycosidic oxygen of lact osethe oxygen that accepts a H atom from Asp59. 1= -70 provides the proper orientation for Asp59 for its interact ion with glycosidic O as well as having a hydrogen-bonding interaction with the guanidini um group of Arg53. Figure 5-24 shows that

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118 Figure 5-21. Histograms of Tyr119 12 populations in MD simulations Dihedral angles are in degrees. Asp59 adopts the proper orientation for catal ysis only in DANA-bound Tr SA, sialyllactosebound TrSA and TrSA5mut simulations. None of TcTS simulations samples this conformation significantly. Additionally, the RMSD change of the loop beari ng Asp59 is investigated. The

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119 Figure 5-22. Backbone RMSD for the loop be aring Tyr119/ Ser119 in MD simulations.

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120 Figure 5-23. Phe58 1 dihedral angle cha nge in MD simulations. Dihedral angles are in degrees. loop architecture change is insignificant (RMSD < 1 data not shown) in all simulations but there is noticeable overall loop motion in only TrSA5mut simulation in which the loop temporarily leaves its original position in the seco nd 10 ns of the simulation (Figure 5-25).

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121 5.3.7.5. Behavior of Tyr342 Dual conform ations of Tyr342 which differ slightly both in 1 and 2 dihedral angles were observed in X-ray crystal st ructures of free TcTS and DANA-bound TcTS. Figure 5-26 and Figure 5-27 shows no sign of dual conformations of Tyr342 in our MD simulations and Tyr342 behaves very similar in all systems. The loop motion is also found to be insignificant since RMSD value of the loop backbone is lower than 1.25 in all systems throughout the simulation (data not shown). Since Ala341 that precedes Tyr 342 in TcTS is replaced by a glycine in TrSA, we also investigated the effect of backbone angle changes. The angle of Ala341 is found to be -50 in all TcTS simulati ons while the homologous Gly341 shows a angle of -130 in all TrSA simulations including TrSA5mut (data not shown). The dihedral angle of Ala341/Gly341 is depicted in Figure 5-28 which shows the dihedral angle changes fr om mostly sampling -150 in free TcTS and TcTS covalent intermediate to mostly sampling -75 in DANA-bound TcTS and sialyllactose-bound TcTS. The same plot shows that the dihedral angle remains the same for all species of TrSA. 5.3.7.6. Behavior of Glu230 Glu230 helps the nucleophile, Tyr342, by captu ring its H before attacking the ligand, thus, its conformation is important. But since Tyr342 conformation and position is found to stay intact in the simulations, the relative position of Glu230 is monitored by the hydrogen bond analysis results and no further analysis is done for Glu230. 5.3.7.7. Behavior of Asp96 Asp96 has dual conformations in DANA-bound TcTS X-ray crystal structure which differ in 1 dihedral angle. Asp96 stays intact in all MD simulations ar ound -70 (data not

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122 shown). Asp96 lies on a -strand whose RMSD is found to be lower than 3 in the simulations, pointing to a mostly stable backbone around this region. 5.3.7.8. Behavior of Leu36 Leu36 has dual conformations in free TcTS and DANA-bound TcTS X-ray crystal structures. S ince Leu36 lies close to Tyr342, the conformation of Leu36 might be affecting Tyr342. Figure 5-29 and Figure 5-30 shows how Leu36 1 and 2 dihedral angles change in the MD simulations. We observe multiple conformati ons of Leu36 in most of the MD simulations. 5.3.8. Ligand-Enzyme Interactions in Ligated TcTS and TrSA MD Simulations In this sec tion, the interactions between th e ligand and the enzyme in different ligated forms of TcTS and TrSA will be compared. Table 5-2 and Table 5-3 gives detailed information about the stability of hydrogen-bondi ng interactions observed in th e catalytic cleft in our MD simulations. Figure 5-31 and Figur e 5-32 illustrate the sialylla ctose-bound forms of TcTS and TrSA, respectively, as an example of the active site interactions of each enzyme. In all ligated TcTS, the carboxyl ate O atoms of sialic acid fo rm salt bridges to Arg314, Arg245 and Arg35 (the arginine triad) guanidinium groups. These constitute the most stable interactions of all. Arg53 guanidinium group forms a hydrogen bond to the hydroxyl group (O4) of the sialic acid for all three cases, except being very scar cely observed in the covalent intermediate. Instead, this O4 atom interacts with the carboxylate O atoms of Asp96 in the covalent intermediate. There are four hydrogen bond inte ractions seen only in si alyl-lactose-bound TcTS. Two of these are intramolecular intera ctions of sialyl-lactoseone betw een the sialic acid O7 atom and the galactose O2 atom and the other between the galactose O5 atom and the glucose O3 atom. It is also observed that Glu362 carboxylat e O atoms form hydrogen bonds to O6 atom of the galactose part of the sialyl-lactose.

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123 Figure 5-24. Asp59 1 change during MD simulations. Di hedral angles are in degrees. In all three ligated TcTS, Asp96 carboxylate O at oms are interacting w ith the N5 atom of the acetyl side chain of the sialic acid.

PAGE 124

124 It is observed that the interactions of the gl ycerol side chain of th e sialic acid differ for each ligated TcTS. The most prominent hydrogen bonds are between Glu230 carboxylate O atoms and O9 atom of the sialic acid in si alyllactose-bound TcTS and between Trp120s side Figure 5-25. RMSD (in ) of the loop beari ng Asp59 in MD simulations without fitting.

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125 Figure 5-26. Histograms of 12 populations of Tyr342 in Tc TS MD simulations. Dihedral angles are in degrees. chain N atom and O8 atom of the sialic acid, and Gln195s sidechain O atom and O9 atom of the sialic acid in the TcTS c ovalent intermediate. In DANAbound TcTS, Asp96 carboxylate atoms interact with both O8 and O9 atoms of the sialic acid as well as Trp120s and Gln195s side chain N atoms interacting with O9 atom. In all ligated TrSA simula tions, the carboxylate group of sialic acid interacts with Arg314, Arg35 and Arg245 (the argi nine triad) guanidinium groups. Additionally, Arg53

PAGE 126

126 guanidinium group and Asp96 carbo xylate group form persistent H-bonds to the hydroxyl group (O4) of the sialic acid. Figure 5-27. Histograms of 12 populations of Tyr342 in TrSA MD simulations. Dihedral angles are in degrees.

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127 Figure 5-28. dihedral angle (in degrees) of Ala341/Gly341 in MD simulations.

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128 Figure 5-29. Histograms of 12 populations of Leu36 in TcTS MD simulations. Dihedral angles are in degrees. The N-acetyl side chain of the sialic acid has its N atom (N5) interacting with the Asp96 carboxylate O atoms in all TrSA simulations. There is an additional interaction seen only in DANA-bound and SLT-bound TrSA si mulations between Asp59s carboxylate O atoms and O atom (O5N) of the acetyl side chain of the sialic acid. This H-bond forms due to a spatial change in Asp59 by changing the 1 angle.

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129 Figure 5-30. Histograms of 12 populations of Ile36 in TrSA MD simulations. Dihedral angles are in degrees. The glycerol side chain of the sialic acid has different interactions in each simulation of ligated TrSA. The most prominent hydrogen bonds lie from Glu230 carboxylate O atoms to O9 atom of the sialic acid in sialyllactose-bound TrSA, and from Trp120s side chain N atom to O8

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130 atom of the sialic acid and from Gln195s sidechain O atom to O9 atom of the sialic acid in the TrSA covalent intermediate. In DANA-bound TrSA Arg245 amino N atoms interact with O9 atom of the sialic acid as well as Trp120s side chain N atom interacting with O8 atom. All these interactions in TrSA simulations are similar to the ones in TcTS simulations except that Asp96 H-bonds to O8 and O9 in DANA-bound TcTS are lost in DANA-bound TrSA. This is due to the different conformations that gly cerol side chain of the sialic acid adopts in the two average structures. Table 5-2. Hydrogen-bond interactions of the ligands in covalent intermediate, DANA-bound and sialyllactose-bound TcTS. Donor Acceptor TcTS-SLT TcTS cov.int. TcTS-DANA 1SA.O1A Arg314.NH1 100 (2900 ps) 96 (260 ps) 92 (121 ps) 1SA.O1A Arg314.NH2 9 (11 ps) 45 (22 ps) 48 (22 ps) 1SA.O1A Arg245.NH1 90 (110 ps) 79 (56 ps) 22 (23 ps) 1SA.O1B Arg314.NH2 99 (1500 ps) 74 (48 ps) 91 (113 ps) 1SA.O1B Arg35.NH1 90 (107 ps) 93 (149 ps) 73 (40 ps) 1SA.O1B Arg35.NH2 65 (34 ps) 47 (22 ps) 79 (51 ps) Asp96.OD1 1SA.O4 42 (45 ps) Asp96.OD2 1SA.O4 19 (23 ps) 39 (37 ps) 1SA.O4 Arg53.NH2 92 (169 ps) 12 (83 ps) 88 (98 ps) Asp96.OD1 1SA.N5 61 (31 ps) 42 (35 ps) 37 (18 ps) Asp96.OD2 1SA.N5 38 (19 ps) 15 (77 ps) 89 (108 ps) Tyr119.OH 1SA.O8 13 (17 ps) Asp96.OD1 1SA.O8 13 (594 ps) 86 (1070 ps) 1SA.O8 Trp120.NE1 45 (76 ps) Asp96.OD1 1SA.O9 75 (814 ps) Glu230.OE2 1SA.O9 16 (24 ps) 9 (46 ps) Glu230.OE1 1SA.O9 88 (230 ps) 14 (261 ps) Gln195.OE1 1SA.O9 32 (71 ps) 12 (154 ps) 1SA.O9 Trp120.NE1 7 (21 ps) 71 (61 ps) 1SA.O9 Gln195.NE2 39 (29 ps) 1SA.O7 3LB.O2 78 (79 ps) 3LB.O5 4GA.O3 70 (38 ps) Glu362.OE2 3LB.O6 48 (72 ps) Glu362.OE1 3LB.O6 19 (46 ps) Percentage occupancies are reported for each interac tion with average lifetimes in parentheses. All ligand atom namings are according to sialyl-lactosebound system. All ligand atoms are colored blue and 4GA, 3LB, and 1SA are the residue numbers of glucose, galactose and sialic acid parts of the sialyl-lactose, respectively.

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131 Figure 5-31. Hydrogen bonding (green dashed lines) and steric (red dashed lines) interactions in sialyllactose-bound TcTS. The lig and sialyllactose is sha dowed and its sialic acid (black), galactose (blue) and glucos e (red) parts are colored separately. There are five hydrogen bond interactions whic h are seen only in sialyl-lactose-bound TrSA. Three of these are intram olecular interactions of sialyl -lactoseone between the sialic acid O5 atom and the galactose O2 atom, anothe r one between the sialic acid O7 atom and the

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132 galactose O2 atom and the third one between th e galactose O5 atom and the glucose O3 atom. The two other H-bonding interactions are observed from the glucose O6 and sialic acid O7 atoms to Ser119 hydroxyl group. It is also observed that Asp362 car boxylate O atoms form hydrogen bonds to O6 atom of the galactose part of the sialyl-lactose and Gln122 side chain forms hydrogen bonds to O1 atom of the glucose part. Table 5-3. H-bond interactions of the liga nds in covalent intermediate, DANA-bound and sialyllactose-bound TrSA. Donor Acceptor TrSA-SLT Tr SA cov.int. TrSA-DANA 1SA.O1A Arg314.NH1 17 (13 ps) 44 (20 ps) 9 (12 ps) 1SA.O1A Arg314.NH2 99 (1600 ps) 94 (198 ps) 97 (406 ps) 1SA.O1A Arg245.NH1 97 (308 ps) 87 (82 ps) 1SA.O1A Arg245.NH2 23 (134 ps) 1SA.O1B Arg314.NH1 98 (522 ps) 84 (71 ps) 98 (474 ps) 1SA.O1B Arg314.NH2 16 (12 ps) 1SA.O1B Arg35.NH2 84 (67 ps) 92 (134 ps) 85 (68 ps) 1SA.O1B Arg35.NH2 45 (25 ps) 79 (50 ps) Asp96.OD1 1SA.O4 99 (1600 ns) 40 (250 ps) 17 (104 ps) Asp96.OD2 1SA.O4 31 (104 ps) 1SA.O4 Arg53.NH2 94 (196 ps) 16 (26 ps) 83 (69 ps) 1SA.O4 Arg53.NH1 26 (341 ps) Asp96.OD2 1SA.N5 94 (168 ps) 46 (101 ps) 53 (32 ps) Asp96.OD1 1SA.N5 10 (25 ps) 70 (53 ps) 1SA.O5N Asp59.OD2 72 (227 ps) 10 (445 ps) 1SA.O8 Trp120.NE1 36 (60 ps) 13 (24 ps) 1SA.O9 Arg245.NH1 23 (55 ps) Glu230.OE1 1SA.O9 92 (1000 ns) 1SA.O9 Trp120.NE1 11 (23 ps) Gln195.OE1 1SA.O9 35 (116 ps) 3LB.O5 4GA.O3 43 (25 ps) 1SA.O5 3LB.O2 10 (13 ps) 1SA.O7 3LB.O2 78 (68 ps) 4GA.O6 Ser119.OG 13 (46 ps) 1SA.O7 Ser119.OG 10 (27 ps) Asp362.OD1 3LB.O6 12 (33 ps) Asp362.OD2 3LB.O6 10 (27 ps) 4GA.O1 Gln122.NE2 24 (30 ps) Gln122.OE1 4GA.O1 9 (30 ps) Percentage occupancies are reported for each interac tion with average lifetimes in parentheses. All ligand atom namings are according to sialyl-lactosebound system. All ligand atoms are colored blue and 4GA, 3LB, and 1SA are the residue numbers of glucose, galactose and sialic acid parts of the sialyl-lactose, respectively.

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133 Figure 5-32. Hydrogen bonding (green dashed lines) and steric (red dashed lines) interactions in sialyllactose-bound TrSA. The lig and sialyllactose is sha dowed and its sialic acid (black), galactose (blue) and glucos e (red) parts are colored separately. 5.3.9. Interactions Observed Between the Enzyme Residu es in the Active Site The hydrogen bonding interactions among the resi dues that lie within 10 of Tyr342s hydroxyl O atom in MD simulations are investigated to detect a ny differences between TcTS and TrSA active sites (Table 5-4 a nd 5-5). Persistent hydrogen bonds are observed in both enzymes

PAGE 134

134 Table 5-4. Hydrogen bonding interactions betw een the residues in the active site in MD simulations of TcTS species. H-bonding atoms TcTS TcTS cov.int. TcTS-DANA TcTS-SLT Asp51.Arg53 OD2-NE: OD1-NH2: OD1-NE: OD2-NH2: 52 (162) 50 (62) 48 (133) 49 (59) 77 (342) 34 (87) 22 (76) 51 (47) 75 (279) 73 (249) 36 (45) 27 (82) 21 (279) 84 (83) 75 (143) Glu230.Arg245 OE1-NH1: OE2-NH1: 39 (80) 60 (121) 99 (1200) 75 (98) 31 (32) 78 (71) Glu357.Arg35 OE1-NE: OE2-NH1: OE1-NH1: OE2-NE: 94 (183) 96 (267) 96 (285) 70 (37) 93 (154) 80 (55) 16 (12) 12 (12) 99 (694) 66 (32) 17 (12) Asp247.Arg245 OD2-NH2: OD1-NH2: OD2-NE: OD1-NE: 53 (31) 60 (35) 40 (53) 51 (58) 47 (59) 45 (57) 44 (41) 45 (38) 56 (31) 55 (32) 50 (52) 49 (49) 61 (53) 42 (34) 62 (69) 36 (44) Glu230.Gln195 OE2-NE2: 86 (513) Gln174.Tyr113 OE1-OH: 80 (4990) 69 (2480) 96 (2650) 96 (463) Glu362.Asn359 OE2-ND2: OE1-ND2: 30 (75) 20 (60) 26 (55) 47 (83) 49 (114) 45 (104) 25 (121) 65 (143) Glu362.Tyr364 OE1-OH: OE2-OH: 42 (80) 34 (77) 17 (47) 36 (53) 48 (96) 40 (79) 24 (78) 65 (138) Glu230.Tyr342 OE1-OH: OE2-OH: 22 (43) 17 (30) 99 (1650) 56 (74) 45 (69) Occupancies are given as percentage and average lif etimes are given in picoseconds in parenthesis. between Asp51 and Arg53, Glu230 and Arg245, Glu357 and Arg35, Asp247 and Arg245, Glu362/Asp362 and Tyr364 side chains. However, TcTS has two additional stable hydrogen bonding interactions between Gln174 and Tyr113, and between Glu362 and Asn359, which are not seen in TrSA due to differences in th e corresponding amino acids (Gln174 in TcTS/Glu174 in TrSA, Glu362 in TcTS/Asp362 in TrSA). In DANA-bound TrSA MD simulation, a unique hydrogen bond between Asp59 and Arg53 is seen. The hydrogen bonding interaction between Tyr 342 and Glu230 is also monitored since these two residues act as a nucleophile couple in the catalytic reactions. In our MD simulations, we found stable hydrogen bonding between these two re sidues only in the c ovalent intermediate forms of the two enzymes and DANA-bound TcTS. A less stable interaction is observed for

PAGE 135

135 unligated TcTS and TrSA. Interestingly, no such interaction is observed in TrSA5mut, either of the sialyllactose-bound enzymes or DANA-bound TrSA MD simulations. Table 5-5. Hydrogen bonding interactions betw een the residues that lie within 10 of Tyr342.OH atom in MD simulations of free and ligated TrSA species. H-bonding atoms TrSA TrSA cov.int. TrSA-DANA TrSA-SLT TrSA5mut Asp51. Arg53 OD2-NE: OD1-NH2: OD1-NE: OD2-NH2: 11 (239) 76 (69) 86 (269) 64 (502) 65 (109) 34 (151) 28 (33) 77 (65) 86 (150) 90 (108) 16 (140) 78 (99) 80 (68) 96 (272) Glu230. Arg245 OE1-NH1: OE2-NH2: OE2-NH1: OE2-NE: OE1-NE: 39 (457) 19 (34) 22 (79) 11 (67) 99 (1600) 11 (64) 86 (291) 48 (27) 49 (203) 49 (210) Glu357. Arg35 OE1-NE: OE2-NH1: OE1-NH1: OE2-NE: 99 (747) 95 (188) 10 (11) 99 (1300) 78 (49) 18 (13) 99 (823) 79 (51) 14 (12) 99 (1380) 63 (28) 15 (12) 94 (202) 94 (158) Asp247. Arg245 OD2-NH2: OD1-NH2: OD2-NE: OD1-NE: 18 (98) 14 (43) 48 (241) 52 (211) 45 (25) 61 (35) 26 (47) 22 (37) 18 (31) 14 (28) 38 (90) 64 (159) 50 (29) 45 (26) 56 (65) 44 (54) 33 (22) 28 (22) Glu230. Gln195 OE2-NE2: OE1-NE2: 40 (246) 34 (337) 10 (451) 89 (1200) 99 (1380) 48 (914) 32 (379) Asp362. Tyr364 OD1-OH: OD2-OH: 92 (192) 49 (82) 22 (72) 48 (161) 43 (158) 44 (167) 42 (139) 26 (174) 66 (204) Glu230. Tyr342 OE1-OH: OE2-OH: 11 (55) 99 (1100) Asp247. Gln283 OD1-NE2: OD2-NE2: 13 (36) 13 (35) Asp59. Arg53 OD1-NH1: 80 (124) Occupancies are given as percentage and average lifetimes are given in picoseconds in parenthesis. 5.4. Discussion 5.4.1. Sialyllactose-Bound Form of TrSA Trp312 form s one of the lateral walls of the catalytic cleft in Tc TS. Experiments proved that Trp312 is crucial for tran s-sialidase activity of TcTS The mutation Trp312-Ala has decreased sialidase activity of TcTS from 306.0 nmol min-1 mg-1 to 185.1 nmol min-1 mg-1 while totally abolishing its trans-sialidas e activity which was 1412.98 nmol min-1 mg-1. Buschiazzo et

PAGE 136

136 al69 also reveals that Pro283-Gln and Tyr248-Gly mutations can abol ish both sialidase and transsialidase activities of TcTS. These mutations are effective on the conformation of Trp312. Proline 283 in TcTS, which is replaced by a glut amine in TrSA, lies righ t next to Trp312 in TcTS and backs the tryptophan that adopts a conf ormation forming a binding site for the sugar donor/acceptor molecules. In the X-ray crystal structure, the glutamine in TrSAthe one that corresponds to Pro283 in TcTSinterferes the sp ace that Trp312 occupies in TcTS, seemingly preventing Trp312 to occupy the same space in TrSA. There is strong experimental evidence pointing to the importance of Pr o283 for trans-sialidase activity: (i) Pro283-Gln mutation abolishing tr ans-sialidase ac tivity of TcTS69 (ii) Chimeric protein that has the first 200 residues from TrSAs C-terminus and the rest from TcTSs N-terminus shows st rict sialidase activity while a single additional Glutamine to Proline (Pro283) mutation can promote both sialidase and trans-sialidase activities.72 In all available TrSA X-ray cr ystal structures, the conformation of Trp312 is different from the one seen in TcTS (Figure 5-33). The two conformations differ both in 1 and 2 dihedral angles and the conformation in TcTS puts the in dole ring of Trp312 in a position to back the lactose part of the sialyllactos e ligand. There is no X-ray crys tal structure of sialyllactose-bound TrSA available to show whether the bindi ng mode of Trp312 to the donor/acceptor lactose molecule is the same as in TcTS or not. Thus, we modeled sial yllactose-bound TrSA by manually placing sialyllactose lig and into the active site of TrSA using DANA-bound TrSA as the starting point and mimicking the sialyllact ose pose in TcTS. The conformational difference between available TcTS and TrSA X-ray crystal structures in Trp312 conf ormation as well as the proven importance of Trp312 and Pro283 for sialyl-t ransfer catalysis draws attention to Trp312. MD simulations of sialyllact ose-bound TrSA model indicated that the loop bearing Trp312 moves away from the sialyllactos e resulting in a slightly more open active site during relaxation

PAGE 137

137 Figure 5-33. Different conformati ons of Trp312 seen in TcTS and TrSA. Sialyllactose is shown in orange. The conformation of Trp312 that ba cks the lactose part of the sialyllactose is seen in all TcTS while Trp312 lies away from the ligand in the other conformation in all available TrSA X -ray crystal structures. of the system. However, in the production run, Trp312 slowly changes its conformation to the proper conformation (1 and 2) observed in sialyllactose-bo und TcTS in about 17 nanoseconds and subsequently re-approaches to the sialyllactose arriving at the exact same binding mode seen in TcTS at the end of 23 ns and conserves this conformation for the rest of the simulation (Figure 5-34). For comparison, the situation in sialyll actose-bound TcTS MD simulation is also shown. The binding mode of Trp312 and the overall loop conformation is conserved throughout the simulation indicating its stabil ity in sialyllactose-bound TcTS. Similar results can also be deduced from the Trp312 1 and 2 histograms in Figure 5-17 and Figure 5-18 which show that Trp312 in TrSA al so visits the dominant conformation seen in TcTS cases, even preferring it over the other conf ormations in some cases. The reason why this conformation is not observed in available X-ray crystal structures of TrSA is in fact found to be related to close crystal contacts. When the unit cell is formed using proper symmetry operations with SwissPdbViewer, the sidechain of Lys495 in the neighboring enzyme swings into the active

PAGE 138

138 site of the enzyme, sterically hindering Trp312 to move freely and adopt the conformation seen in TcTS (Figure 5-35). The conformation seen in TrSA X-ray crystal st ructures causes hydrogen Figure 5-34. Behavior of Trp312 and its loop ba ckbone in MD simulations of A) modeled sialyllactose-bound TrSA and B) sialyl lactose-bound TcTS. The loop of Trp312 approaches to the sialyllactose lig and and Trp312 adopts the binding mode conformation during simulation in TrSA while the loop of Trp312 and binding mode conformation remains stable duri ng the whole 50-ns simulation.

PAGE 139

139 bonding interactions between Trp312s indole N atom and Asp362s carboxylate O atoms and between Lys495s side chain N at om and Tyr364s phenol O atom. All the above results in addition to the MD simulation of TrSA with manually placed sialyllactose point to a unique Trp312 conformation both in TcTS and TrSA forming the binding site for the lactose moiety. This unique confor mation is the Trp312 conformation seen in all free and ligated TcTS X-ray structures. Tyr119 constitutes the second late ral wall of the catalytic cl eft facing the lateral wall constituted by Trp312 and is also found to be very significant for the tran s-sialidase catalytic Figure 5-35. Close crystal contact s in the unit cell of unligated TrSA. Orange and blue ribbons show the two neighboring enzymes. Lys495 side chain lies inside the active site of TrSA hindering Trp312 to move freely. Ser119 is also show n to clarify the relative orientation. ability of TcTS. The mutation Tyr119Ser causes th e trans-sialidase activity to decrease to 2.5 % of that of the wild type TcTS while the sialidas e activity only decreases to 77.8% of that of the wild type TcTS (Table 4 in Paris et al48). In place of Tyr119 in TcTS a serine exists in TrSA. The mutation of this serine in TrSA to a tyrosine is unable to promote a ny trans-sialidase activity as well as decreasing the sialidas e activity to 50 % compared to th e wild type (Table 3 in Paris et al48). These data were initially interpreted that two different binding sites exist for the acceptor

PAGE 140

140 and the donor lactose molecules. However, the X -ray crystal structures elucidated later showed that the active sites of TcTS and TrSA were not large enough to accommodate the donor and acceptor at the same time. While comparing the average structures of sialyllactose-bound TcTS and TrSA produced by MD simulations, we noticed a structural differen ce that can shed light on this issue. Phe58 in TcTS, which is also present in TrSA, adopts diff erent conformations in the two enzymes. In TcTS, Phe58 leans back ( 1=60 or -60) and the lactose part of the ligand is embraced from the two sides by Trp312 and Tyr119. However in TrSA, Phe58 adopts to a conformation ( 1= 180)which is different from th e starting conformationsuch that it embraces the lactose part of the ligand from the opposite side of Trp312 (F igure 5-36). Thus, the absence of Tyr119 in TrSA is covered by the presence of Phe58, complementing the binding site formed by Trp312 on one side. Still, the data imply that the lactose should be delicately oriented for trans-sialidase catalysis to happen. A shift in orientation of the lact ose caused by interacti ng with Phe58 instead of Tyr119 can significantly decreas e the trans-sialidase activity. Figure 5-23 shows that in sialyllactose-bound TrSA model, Phe58 1 dihedral angle adopts a binding mode different than th e initial conformation which was the same as the conformation seen in sialyllactose-bound TcTS and keeps this conformation for th e rest of the simulation. This binding conformation is depicted in the averag e structure in Figure 536 for sialyllactose-bound TrSA. In fact, this phenylalanine which exists in both TcTS and TrSA X-ray crystal structures is not a natural residue for TcTS which indeed has an asparagine residue instead.75 This Phe58 is one of the 7 mutations performed to obtain stable diffraction-quality crystals (All the other 6 mutations are in the lectin-like part.). Thus, th e presence of Phe58 might be playing a role in

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141 sialidase action in TrSA, helpi ng it to bind the lactose part of the ligandtaking the role of Tyr119 in TcTS. Table 5-6 give s the sequence information for si alidases and trans-sialidases from Trypanosoma cruzi, brucei and rangeli. We see a phenyleither right before or right afteraround Asp59 homologs in each sialidase while th e sequence differs in trans-sialidases. Thus, presence of the Phe58 homolog s next to the acid/base catalyst aspartic acid residue could be a signature for trypanosomal sialidases. Figure 5-36. Binding mode of si alyllactose in A) TcTS and B) TrSA. Trp312 and Tyr119 are embracing lactose part of the ligand in TcTS. Trp312 and Phe58 are embracing lactose part of the ligand in TrSA which has a serine in place of Tyr119. Table 5-6. Sequence information for differ ent sialidases and trans-sialidases of Trypanosomal species. Source Enzyme PdbID/Seque nceID Sequence around Asp59 analogs Acid/base catalyst T.cruzi trans-sialidase 1MS3 N D N Asp59 T.rangeli sialidase 1N1S F D N Asp63 T.brucei sialidase Q57YT6-1 E D F Asp108 T.brucei trans-sialidase Q57XJ2 T D Y Asp157 T.brucei trans-sialidase Q9GSF0 T D Y Asp157 The average sialyllactose pose is found to be very similar in sialyllactose-bound TcTS and TrSA MD simulations. However, in the active sites, the conformation of Asp59 differs for the two enzymes; Asp59 adopts a proper conformation for catalysis in sialyllactose-bound TrSA in contrast to sialyllactos e-bound TcTS. There is also a slight difference in the loop bearing Tyr342 A B

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142 which might be related to the presence of Ala 341 in TcTS instead of Gly341 in TrSA. There are also several mutations that are constraining the active sites differently: (i) Val95 in TcTS is Met95 in TrSA which re sults in a more restricted active site in TrSA around 1SA.O4. (ii) Leu176 in TcTS is Val176 in TrSA which results in a more restricted active site in TcTS around N-acetyl group of sialic acid. Comparing the ligand-enzyme interactions, one notices the stable hydrogen-bonding of 1SA.O4 with Asp96 carboxylate O atoms in TrSA is much less in TcTS and the one between 1SA.O5N and Asp59 carboxylic acid O atoms in TrSA is absent in TcTS due to Asp59 conformational change (Table 5-2, Table 5-3). Additionally, Gln122 in TrSA which corresponds to a serine residue in TcTS interacts with 4 GA.O1 atom of the sialyllactose. The RMSF analysis results depicted in Figure 5-10 show no sign of a big difference in the active sites except for the loop bearing Trp312wh ich is a sign of this loop rearranging for proper ligand binding in TrSAa nd Ala341 which lies right next to catalytic Tyr342. The absence of Tyr342 conformational change as seen in the histograms in Figure 5-26 and Figure 527 and the higher conformati onal flexibility seen in dihedral angle of Ala341 in TcTS compared to Gly341 in TrSA (Figure 5-28) may be a sign of the effect of Ala341 flexibility on catalysis in TcTS. In summary, the sialyllactose binding to TrSA is found to be very similar to the case in TcTS except for that Phe58 and Gln122 residues help binding on one side of the catalytic cleft in TrSA instead of Tyr119 residue in TcTS. 5.4.2. Effects of the 5 Point Mutations on TrSA that Promote trans-Sialidase C atalysis Since only 5 point mutations (Met95Val, Ala97Pro, Ser119Tyr, Gly248Tyr, Gln283Pro) were enough to modify the strict sialidase TrSA into a trans-sialid ase, the effects of these 5 point mutations will be disccussed here.

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143 Due to lower resolution, the B-factors of TrSA5mut X-ray crystal structure are found to be incomparable with the wild-type TcTS and TrSA as depicted in Figure 5-15. The RMSF results of MD simulations are thus used to find the differences in flexibilities of the residues of each system. Figure 5-9 showed that the flexibilit ies in the loops of Ser119, Gly248 and Gln283 in TrSA have decreased due to the mutations, approaching to the fl exibilities of homologous loops in TcTS. It is also important that in TcTS, the flexibilities of Tyr248 and Pro283 are still lower compared to TrSA despite the loop opening observed in Trp312 loop of TcTSwhich gives these two neighboring loops more degrees of free dom. These 3 loops are important in forming the binding site for the lactose part of sialyllact ose ligand in TcTS; Tyr119 forms one lateral wall of the binding site for lactose and Trp312 forms the second lateral wall which is supported firmly by Tyr248 and Pro283 by hydrophobic interactions. RMSF results confirm that the mutations of Gly248Tyr and Gln283Pro in TrSA provide d a stable support for Trp312. However, we observed that Trp312 did not sample the bindi ng conformation (Figure 5-17) in the time scale of our TrSA5mut MD simulation. The initial conformation of Trp312 in TrSA5mut is different from the binding conformation due to crystallographic constr aints as explained for TrSA above and affects our ability to sample the binding conformation. However, combining all information obtained from MD simulations of TcTS and TrSA, the loop of Trp312 is able to form a more open active site espe cially in the absence of the stacking interaction with Tyr119 due to Tyr 119 side chain swinging down in Tc TS or a serine replacing Tyr119 in TrSAand once the loop opens, Trp312 can sample all pos sible conformations. Especially the free enzymeswild-type TcTS, wild-type TrSA and TrSA5mutare observed to move more freely. Introduction of sialyllactose limits this degree of freedom and locks Trp312 into proper conformation as we have seen in MD simula tion of modeled sialylla ctose-bound TrSA. Figure 5-

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144 17 also indicates the stability of binding conformation of Trp312 in TcTS once the sialic acid binding region is filled while no similar beha vior is seen in TrSA. Whether the binding conformation of Trp312 in TrSA is not stable due to lack of hydrophobic interactions or the simulation was not long enough to sample all co nformational space is not certain. Either the energetics of this conformational change should be elucidated or more MD simulations with different initial conformations of Trp312 should be performed to gi ve a certain answer to this question. The other two mutations Met95V al and Ala97Pro did not change the flexibility of the strand bearing these or the conformation of Asp96 to a noticable degree. However, the replacement of methionine with a less bulky re sidue of valine decreases the constraints around O4 of sialic acid and affects the hydr ogen bonding network in that region. 5.4.3. Comparison of DANA-Bou nd Forms of TcTS and TrSA DANA, which is a very effective inhibitor for sialidases and specificly for TrSA, was unsuccessful in inhibiting TcTS. We investigated the structure and dynamics of the enzymes to find the reason of this inhibition difference. The main difference between DANA-bound forms of TcTS and TrSA is that the glycerol branch of DANA adopts different co nformations in the two enzymes (Figure 5-37) which is also seen between the X-ray crystal structures. In DANA-bound TcTS the glycerol branch of DANA leans downwards interacting with both Glu230 and Gln195 and forcing Glu230 to move towards Tyr342 and make a hydrogen-bonding interaction that is very stable throughout the simulation. However, in DANA-bound TrSA, the glycerol branch lies straight leavin g the lower cavity for Glu230 which is now leaned away from Tyr342 and bended towards Gln195 having a very stable hydrogen-bonding interaction with it. Due to th is conformation differe nce, the hydrogen bonding interactions of the glycerol branch with Trp120 indole ring an d Asp96 carboxylate group seen in

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145 DANA-bound TcTS are also lost in DANA-bound TrSA. No such difference in Glu230 conformations is observed in the X-ray crystal structures. One could argue that different conformations of the glycerol branch of DANA seen in the average structures might be due to the different conformations of them in the X-ray crystal structures that are used as st arting structures since these conformations are preserved throughout the simulation mainly. Whether there are at leas t two conformational minima for the glycerol Figure 5-37. A view from superimposed DANAbound forms of TrSA (orange) and TcTS (gray) average structures of the MD simulations. DANA is s hown in licorice form and Tyr342, Glu230 and Gln195 are shown in CPK form. The conformation of glycerol side chain of DANA differs in the two cas es also affecting Glu230 conformation. branch of DANA in both TcTS and TrSAthat we could sample only one in each of our simulationsor there is really one single minimum for each enzyme which constitutes a real difference in DANA binding of TcTS and TrSA is a question. The simulations of TcTS covalent intermediate and sialyllactose-bound TcTS show that the glycerol branch of these ligands adopt the conformation seen in DANA-bound TrSA and do not even sample the conformation seen in DANA-bound TcTS. Since all these ligands occupy th e same space in TcTS in the sialic acid binding site, it is more likely that there are at least two different stable conformations for the Tyr342 DANA Glu230 Gln195

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146 glycerol branch of DANA (and si alic acid) in TcTS. In fact, dua l conformations of the glycerol side chain are observed in the X-ray crys tal structure of DANA-bound TcTS. Thus, the conformation of this glycerol branch needs further investigation. The Asp59 conformations are also differen t in the average structures of DANA-bound TcTS and TrSA (Figure 5-38) although there is no such difference in the original X-ray crystal structures of these two enzymes. The conformation in the crystal structures is the same as the one seen in the average structure of DANA-bound Tr SA in which Asp59 leans towards DANAas it Figure 5-38. A view from superimposed DANAbound TrSA (orange) and TcTS (gray) average structures of the simulations. DANA is shown in licorice form and Asp59, Met95/Val95 and Arg53 are shown in CPK form (with no H atoms shown). Asp59 is affected from the bulky residue Met95 in TrSA compared to Val95 in TcTS. The conformation of Asp59 is also reflected in stable hydrogen-bonding interaction of Asp59 with Arg53 in TrSA which is seen in TcTS. would do in the presence of the glycosidic O of the sialyllactose that accepts a proton from Asp59 in the proposed mechanism. Investigatin g the crystal contacts of DANA-bound TcTS and DANA-bound TrSA reveals that ther e are two different crystallogr aphic constraints forcing the same conformation of Asp59: (i) The unit cell obtained from X-ray crys tal structure of DANA-bound TrSA shows that Asp59 is interacting with the Lys 495 of the neighboring enzyme (shown in Figure 5-35) through a wate r-mediated hydrogen bond. Asp59 Met95/Val95 DANA Arg53

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147 (ii) The X-ray crystal structure of DANA-bound TcTS includes a solvent glycerol molecule that locates itself into the ac tive site occupying the same position that would be occupied by galactose part of the lactose molecule. The hydrogen bonding interaction between one hydroxyl gr oup of this glycerol molecule and Asp59 promotes a certain Asp59 conformation. Due to these two constraints found, the Asp59 conf ormations in X-ray crys tal structures are not reliable or natural. When the 1 dihedral angle of Asp59wh ich is the source of the conformation differenceis followed over th e simulation time, each of the two DANA-bound enzymes is found to adopt mostly one single conformation that is different for each case (Figure 5-24). Different Asp59 conformations can be rela ted to the neighboring bu lky residue Met95 in TrSA occupying more space compared to the corresponding residue of Val95 in TcTS. The Asp59 conformation difference is also reflec ted on that hydrogen bonding between Asp59 and Arg53 is observed 80% of the simulation in DANA-bound TrSA compared to 3% in DANAbound TcTS. Another difference between DANA-bound forms of TcTS and TrSA are in hydrogen bonding interactions of Asp96. Asp96 interacts with O4 of DANA in DANA-bound TcTS MD simulation, but no such interaction is obser ved in DANA-bound TrSA si mulation, in which Asp96 interacts with the last hydroxyl group on the glycerol branch of DANA instead. Since the position and conformation of Asp96 is found to stay intact in MD simulations, a slight conformational change of DANA must exist between the two enzymes. In DANA-bound TrSA, DANA is tilted slightly down on the side of O4 hydroxyl group to interact with Asp96 while in DANA-bound TcTS, the glycerol branch of DANA bends downwards to interact with Asp96. Trp312 did not sample the binding conformation in DANA-bound TrSA MD simulation as seen in the histograms of Figure 5-17 in c ontrast to DANA-bound TcTS (Figure 5-18). Since Trp312 is found to retain its init ial conformation mainly, it is ha rd to comment on the possible

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148 effect of DANA binding on Trp312 conformation. RMSF analysis shows that the loops of Gly248/Tyr248 and Trp312 are more flexible in DANA-bound TrSA compared to DANA-bound TcTS (Figure 5-7 and 5-8). As mentioned before, the presence of Tyr248 and Pro283 promotes the binding mode of Trp312 and de creases the flexibilities of thes e loops in TcTS compared to TrSA. The slight difference in the positioning of D ANA and the mentioned differences in several hydrogen bonding interactions between DANA-b ound forms of TcTS and TrSA might be responsible for the signifi cant difference in their Ki values. More simulations are necessary to confirm these observations. The dual conformati ons of the glycerol branch of DANA in DANAbound TcTS crystal structure also require more attention and follow up. A new simulation of DANA-bound TcTS starting with the alternative c onformation of the glycerol branch of DANA can shed some light. 5.4.4. Comparison of Unligated and DANA-Bound Forms of TcTS Surface plas mon resonance studies75 mentioned in the Introduction clearly showed that lactose could not bind TcTS in the absence of sia lic acid or sialyllactose in the medium and there is no known reason for this observation. Several di fferent studies also showed that TcTS can produce DANA as a by-product.49,75 Combining these data, we can hypothesize that TcTS might be producing DANA using sialic acid or sialylla ctose in the medium wh ich subsequently causes some modifications in TcTS to promote lactose binding. In our simulations, we observed that DANA binding causes a decrease in the mobilities of the three loops bearing Tyr248, Pro283 and Trp312 (Figure 5-7 and 5-8). The lower mobilities of these three loops will mean a more rigid agly con binding site readily available for lactose binding which can explain the observed change of lactose binding ability of TcTS in the presence of sialic acid or sialyllactose in the medium.

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149 5.4.5. Comparison of the Effect of Covalent Intermediate Formation in TcTS and TrSA We have noticed a difference between TcTS and TrSA in the effect of covalent interm ediate formation. As shown in Figure 5-19 the loop bearing Trp312, which resides at the periphery of the catalytic cleft, is observed to move outwards and form a more open active site in unligated TcTS and covalent intermediate of TrSA. However, no such motion is observed in unligated TrSA and covalent intermediate of TcTS. It is known that TcTS can catalyze both hydr olysis and sialyl-tra nsfer reactions, while TrSA can only catalyze hydrolysis. The ability of Tc TS to protect the covalent intermediate from the attack of surrounding water molecules until an acceptor molecule binds and completes the transfer reaction is of curiosity. Since in TrSA, a very similar active site the covalent intermediate readily reacts with the water molecule s, it is possible that solvent exposure of the catalytic active sites have a role. The MD simulation results in Figure 5-19 mi ght be pointing to the change of solvent exposure of the active sites differ ently in the two enzymes in the course of the reaction. TcTS active site is solvent-exposed in the unligated form but once the covalent intermediate forms, solvent exposure is limited by the closure of the loop bearing Trp312. This will help TcTS to protect the covalent intermediate from the inte nsive attack of water molecules. However, in TrSA, the active site becomes more solvent-exposed after the covalent intermediate formation which is in line with its ve ry high hydrolysis efficiency and no sialyl-transfer ability. Although there might be other structural, energetical or dynamical reasons, or a combination of those for the observed catalyt ical difference of TcTS and TrSA, water accessibility also stands as a good candidate for such an outcome and requires further investigation. More simulations, with different starting conformations of Trp312 to eliminate its

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150 possible effect in the observed loop opening difference, should be performed to test this hypothesis.

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151 CHAPTER 6 CONCLUSIONS AND PERSPECTIVE Trans-s ialidase (TcTS) facilitates Trypanosoma cruzi, the parasite responsible for Chagas disease, to evade from the host immune respons e and to invade the hos t cells and thus, is identified as an appealing therapeutic target for uncurable Chagas diseasewhich is a drastic threat to large human populations in Central and South America. TcTS catalyzes the transfer of sialic acidswhich is used by the host immune sy stem to distinguish host cells from others and can not be synthesized de novo by T.cruzifrom host glycoconjugates to the parasites glycoconjugates. The mechanism of TcTS has been the subject of various research efforts which aim to design potential inhibitors. Trypanosoma rangeli sialidase (TrSA), which acts as a strict sialidase despite its distinct structural si milarity to TcTS, pointed out that minute structural modifications are responsible for trans-sialidase catalytic ability. T hus, TrSA is used as a case study together with TcTS to elucidate the requirements for achieving trans-sialidas e catalysis. Continuous efforts recently succeeded in transforming TrSA into a trans-sialidase with only 5 point mutations at the active site. The results from kinetic isotope effect studies together with chemical trapping experiments for TcTS anticipated that an associative mechanis m is active in the first step of TcTS catalytic reactionwhich is scavenging the sialic acid fr om sialyllactosewith significant nucleophilic participation which results in collapsing into a covalent intermediate. The ping-pong mechanism implicated by these results was indeed ruled out mistakenly due to earlier intial velocity studies. X-ray crystal structures of different forms of TcTS obtained la ter also started a debate about the identity of residues that act as nucleophile and acid/base cataly st since the candidates for these roles according to their lo cation were very unusual.

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152 This study started while the mechanism and the identity of th e nucleophile and the acid/base catalyst were still being debated. We approached the open-ended questions in this topic with a computational chemists point of view. Mo re experimental results became available in the meanwhile shedding light on our path. Evidence fo r covalent intermediate formation in TrSA using an activated ligandwhich might possibly be biased by the artificial substituents of the liganddirected us to also investigate the possibility of covalent intermediate formation in TrSA. Our QM/MM studies with two separate methods have confirmed energetic plausibility of covalent intermediate formation in both TcTS and TrSA with natural ligands using Tyr342/Glu230 couple as the nucleophile and Asp59 as the acid/base catalyst. Potential energy surfaces constructed for TcTS are in line with the experimental da ta showing that TcTS acts as a better trans-sialidase than a sialidase in the presence of acceptor glycoconjugates. Our calculations have identified the oxocar benium ion form of sialic acid to be an intermediate rather than a transition structure. However, the energy barrier calculated for the oxocarbenium ion to convert into the covalent intermed iate is low. Scavenging the sia lic acid from sialyllactose (i.e. the formation of oxocarbenium ion) is determined to be the rate-determining step that has a late oxocarbenium-like transition structure. Due to the relative timing of bond cleavages/ formations and the formation of a stable ionic intermediate on the reaction path, we can describe the TcTS mechanism as SN1-like although it includes significant nuc leophilic participation and is more complex than a simple SN1 mechanism. Molecular dynamics simulations we performed, on the other hand, shed some light on the path to be followed to elucidate the inhibitor binding differences between TcTS and TrSA. The glycerol side chain of sialic acid is found to be a good candidate to adopt different conformations

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153 in the two enzymes, thereby causing different inhibitor binding abilities. The simulations also showed that the conformation of Trp312 in the aglycon binding sites we re biased by crystal contacts in the X-ray crystal structures of TrSA, and Trp312 indeed adopts the same conformation in TrSA as in TcTS. However, stil l a difference exists between TcTS and TrSA in that Trp312 is found to be more flexible in TrSA, most probably related to the replacement of Tyr119 in TcTS by a serine residue. Additiona lly, the flexible loop bearing Trp312 which is found in a specific conformation in all X-ray crysta l structures proved to be artificial and the movement of this loop has a possible role in th e binding affinity of the enzyme to its ligands. This finding is also an indicati on of the fact that considering the X-ray crystal structures for direct use in ligand docking studies can mislead the researchers, especially in the case of enzymes such as TcTS and TrSA for which the ligand binding site mostly consists of flexible loops.

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164 BIOGRAPHICAL SKETCH zlem Demir got her BS degree in 2000 and he r Master of Science degree in 2002 from the Chemistry Department of Bilkent University in Turkey. She then came to University of Florida to pursue PhD in computational chemistry.