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1 CARBONIC ANHYDRASE EXPRESSED IN THIOMICROSPIRA CRUNOGENA GAMMAPROTEOBACTERIUM By NATALIA ADRIANA DIAZ TORRES A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DE GREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012
2 2012 Natalia Adriana Daz Torres
3 To my amazing parents, Jos A. Daz Escribano and Virgen M. Torres Daz
4 ACKNOWLEDGMENTS I am eternally grateful for working with a most wonderful mentor, Dr. Robert McKenna, whose guidance and advice, as well as his sense of humor, helped me to achieve my best when I was feeling my worst. I thank him greatly for his understand ing and his willingness to listen and help in any way he can. I doubt that I will ever be able to work with a superior as great as him. I extend my thanks to all those who helped me complete my project. Dr. David Silverman was very helpful and also very attentive to my progress. Dr. Chingkuang Tu helped me conduct some experiments which were crucial to the completion of my project. Dr. Lin da Bloom was a part of my defense committee and helped me understand what was necessary to complete the requirements to obtain my graduate degree. Without them, I would not have been able to get where I am today. I would also like to thank all of my labora tory research fellows (lab mates) for all their advice. Particularly, I would like to thank Mayank Aggarwal, Chris Boone, and Dr. Balasubramanian Venkatakrishnan, for having the patience to deal with me on a daily basis and help me out in any way they coul d. I would also like to thank all the folks at the virus lab, Dr. Mavis Abgandje McKenna included, for all of their help and advice at the joint group meetings which helped me grow as a researcher and a presenter. I would like to thank my parents Jos A. Daz Escribano and Virgen M. Torres Daz, and my brother Ricardito, for their unconditional love and support. It was great to have them at my back, cheering me on no matter the circumstances. And lastly, I would like to thank my fianc, Juan Carlos Castill o, without whom I would not have been able to carry forward with this project. My time at the University of Florida has been filled with many great memories that I will cherish wherever I go.
5 TABLE OF CONTENTS page ACKNOWLE DGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 6 LIST OF FIGURES ................................ ................................ ................................ .......... 7 LIST OF ABBREVIATIONS ................................ ................................ ............................. 9 ABSTRACT ................................ ................................ ................................ ................... 14 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 16 Classification of CAs ................................ ................................ ............................... 16 Cataly tic Mechanism of CAs ................................ ................................ ................... 19 Chemolithoautotrophs and Thiomicrospira crunogena ................................ ........... 21 The T. cru CA ................................ ................................ ................................ ...... 23 2 MATERIALS AND METHODS ................................ ................................ ................ 32 Expression and Purification of T. crunogena CA ................................ ................. 32 Gel Filtration Chromatography ................................ ................................ ................ 33 Crystallization and X ray Data Collection of T. cru CA ................................ ........ 33 Structure Determination of T. cru CA ................................ ................................ ... 34 Oxygen 18 Exchange Kinetic Analysis ................................ ................................ ... 35 Differential Scanning Calorimetry (DSC) ................................ ................................ 37 3 RESULTS ................................ ................................ ................................ ............... 38 4 DISCUSSION ................................ ................................ ................................ ......... 59 Crystal Structure of T. cru CA ................................ ................................ .............. 59 The N terminus of T. cru CA ................................ ................................ ................ 64 The Disulfide Bond ................................ ................................ ................................ 65 The Dimeric Interface ................................ ................................ .............................. 68 5 CONCLUSIONS ................................ ................................ ................................ ..... 95 LIST OF REFERENCES ................................ ................................ ............................... 98 BIOGRAPHIC AL SKETCH ................................ ................................ .......................... 108
6 LIST OF TABLES Table page 3 1 Data collection and refinement statistics for the crystallographic study of T. cru CA. ................................ ................................ ................................ ............ 45 3 2 Comparison of maximal (pH independ ent) catalytic parameters for T. cru CA and hCA II. ................................ ................................ ................................ .... 46 3 3 Comparison of inhibition constants for T. cru CA and hCA II. ......................... 47 3 4 Thermodynamic parameters of unfolding of T. cru CA at different pH values. ................................ ................................ ................................ ................ 48 4 1 Possible interfaces observed in the crystal structure of T. cru CA as determined by PISA (102) ................................ ................................ .................. 74 4 2 Hydrogen bond interactions at the T. cru CA dimer interface. ......................... 75 4 3 Comparison of the interfaces of T. cru CAs ............... 76 4 4 Comparison of dimer interfaces by secondary structure matching (SSM) superposition of T. cru CA using Coot (64) ................................ ..................... 77 4 5 CAs ................................ ...................... 78
7 LIST OF FIGURES Figure page 1 1 Cartoon representation of hCA II. ................................ ................................ ....... 25 1 2 Cartoon representation of hCA II active s ite residues. ................................ ........ 26 1 3 CA expressed in Haemophilus influenzae bacterium ................................ ................................ ................................ ........... 27 1 4 CA expressed in the thermophile Methanosarcina thermophila ................................ ................................ ............. 28 1 5 Hydrogen bonding network involved in the zin c hydroxide mechanism of hCA II. ................................ ................................ ................................ ........................ 29 1 6 Transmission electron micrograph of Thiomicrospira crunogena XCL 2 cells ... 30 1 7 Putative model for the interaction of four carbo nic anhydrases in the T. cru gammaproteobacterium. ................................ ................................ ..................... 31 3 1 Crystals of T. cru CA. ................................ ................................ ...................... 49 3 2 Crystal structure of T. cru CA. ................................ ................................ ......... 50 3 3 Superposition of the T. cru CA monomer and the hCA II crystal structure. ..... 51 3 4 Close up stick representation of superimposed active site residues in T. cru CA and hCA II ................................ ................................ ................................ 52 3 5 The pH profiles for R 1 for the hydration of CO 2 catalyzed by T. cru CA and hCA II ................................ ................................ ................................ ................. 53 3 6 Th e pH profiles for k cat exch /K eff CO2 (M 1 s 1 ) for the hydration of CO 2 catalyzed by T. cru CA and hCA II ................................ ................................ ................. 54 3 7 The pH profile for R H2O /[E] (s 1 ) for proton transfer in the dehydration direction catalyzed by T. cru CA and hCA II. ................................ ................... 55 3 8 The inhibition profiles of T. cru CA by iodide, chloride, and bromide. .............. 56 3 9 The pH pro file of the melting temperature of the dissociation of dimeric T. cru CA.. ................................ ................................ ................................ ................. 57 3 10 The pH profile of the melting temperature for the unfolding of dimeric T. cru CA ................................ ................................ ................................ .................. 58 4 1 Representation of the crystal packing of T. cru CA. ................................ ........ 79
8 4 2 Ramachandran plot of T. cru CA residues obtained from PROCHECK ......... 80 4 3 Cartoon representation of T. cru CA ................................ ............................... 81 4 4 Close up stick representation of the T. cru CA active site. .............................. 82 4 5 Comparison of T. cru CA and hCA II ................................ .............................. 83 4 6 Sequence alignment of hCA II and T. cru CA determined by ClustalW .......... 84 4 7 Disorder probabil ity plot obtained from PONDR FIT ................................ .......... 85 4 8 Cartoon representation of the dimer formed by chains A and B of T. cru CA. ................................ ................................ ................................ ..................... 86 4 9 Possible interfaces present at the crystal structure of T. cru CA .................... 87 4 10 Buried lysines at the T. cru CA dimer interface. ................................ .............. 88 4 11 CAs T. cru CA and hCA VI. ............................. 89 4 12 Superimposition of T. cru CA and hCA IX. ................................ ...................... 90 4 13 Superimposition of T. cru CA and hCA XII. ................................ ..................... 91 4 1 4 Superposition of T. cru CA and Cr ................................ ....................... 92 4 15 Superimposition of T. cru CA and Ao CA. ................................ ........................ 93 4 16 CAs on T. cru CA. ................................ ........ 94
9 LIST OF ABBREVIATION S Angstrom A CA P Rhodopseudomonas palustris CA A O CA Aspergillus oryzae carbonic anhydrase A RG Arginine A SN Asparagine A SP Aspartate AU Arbitrary unit AU Asymmetric unit B Base B FACTOR Temperature factor C C elsius CA Carbonic anhydrase C A 2+ Calcium CCD Charge coupled detector CCM Carbon concentrating mechanism CM centimeter CO 2 carbon dioxide C R CA1 Chlamydomonas reinhardtii carbonic anhydrase C SO CA carbonic anhydrase CSS Complexation significance score CV Column volume C YS Cysteine 3 D Three dimensional D A Dalton
10 DIC Dissolved inorganic carbon DNA Deoxyribonucleic acid DSC Differential scanning calorimetry E Enzyme E. COLI Escherichia coli F C Calculated structure factor F O Observed structure factor FPLC Fast protein liquid chromatography G DN HC L Guanidine hydrochloride G LN Glutamine G LU Glutamate G LY Glycine G3P Glyceraldehyde 3 phosphate GPI Glycophsph atidylinositol H Hour enthalpy V H CA II H uman carbonic anhydrase isoform II H CA IV H uman carbonic anhydrase isoform IV H CA VI H uman carbonic anhydrase isoform VI H CA IX H uman carbonic anhydrase isoform IX H CA XII H uman carbonic anhydrase isoform XII HC L Hydrochloric acid HCO 3 B icarbonate
11 HEPES 4 (2 hydroxyethyl) 1 piperazinesulfonic acid H IS Histidine H 2 O Water HS Hydrogen sulfide I LE Isoleucine IPTG Isopropyl D galactopyranoside K Potassium K B C atalytic turnover for proton transfer K CAT C atalytic turnover rate K CAT /K M Catalytic efficiency K D A K ilodalton K EFF CO2 A pparent affinity constant of CO2 to carbonic anhydrase L EU leucine L I + Lithium L YS L ysine M Molar M 2+ Bivalent metal ion M ET Methionine MG M illigram M G 2+ Magnesium MIN Minute MM Millimeter M M Millimolar MR Molecular Replacement M. THERMOPHILA Methanosarcina thermophila
12 G M icrogram S M icrosecond N 2 Nitrogen N A C L Sodium chloride N A 2 SO 4 S odium sulfate NCBI National Center for Biotechnology I nformation NGCA Neisseria gonorrhaea carbonic anhydrase NM N anometer 18 O O xygen 18 OD O ptical density PAGE P olyacrylamide gel electrophoresis P AMBS p (aminomethyl)benzenesulfonamide PDB Protein Data Bank PEG P olyethylene glycol PGA P hosphoglyceric acid P H Negative decimal logarit hm of the hydrogen ion activity in a solution P HE P henylalanine PISA Protein Interfaces, Surfaces, and Assemblies P K A A cid dissociation constant P RO P roline PSR P roton shuttle residue R C orrelation coefficient R U B IS CO Ri bulose 1,5 bisphospate carboxylase o xygenase RMSD R oot mean square deviation R. PALUSTRIS Rhodopseudomonas palustris
13 RPM R evolutions per minute S Second SDS S odium dodecyl sulfate S ER S erine S R 2+ S trontium SUMO S mall ubiquitin related modifier TCA tricarboxylic acid TCEP tris(2 carboxyethyl) phosphine T. CRU Thiomicrospira crunogena T HR T hreonine T M M elting temperature T RIS T ris(hydroxymethyl)aminomethane T RP Tryptophan T YR T yrosine UV U ltraviolet region of electromagnetic spectrum V AL V aline V IS V isible region of electromagnetic spectrum V M 2XYT 2X Yeast extract and tryptone Z N 2+ Z inc
14 Abstract Of Thesis Presented To The Graduate School Of The University Of Florida In Partial Fulfillment Of The Requirements For The Degree Of Master Of Science STRUCTU RAL, KINETIC, AND BIOP HYSICAL CHARACTERIZATION OF AN CARBONIC ANHYDRASE EXPRESSED IN THIOMICROSPIRA CRUNOGENA GAMMAPROTEOBACTERIUM By Natalia Adriana Daz Torres August 2012 Chair: Robert McKenna Major: Biochemistry and Molecular Biology Carbonic anhydrases (CA) are mainly zinc metalloenzymes that catalyze the interconversion of water and carbon dioxide to bicarbonate and protons. As such, CAs are involved in many important physiological processes, such as respiration and pH regulation in tissues, making them essential to most living organisms. Thiomicrospira crunogena XCL 2 ( T. cru ) is a novel sulfur oxidizing chemolithoautotroph that plays a significant role in the sustainability of deep sea hydrothermal vent communities. The recently dis covered deep sea vent gammaproteobacterium, Thiomicrospira crunogena XCL 2, encodes and expresses four different CAs from all evolutionarily and structurally distinct families of carbonic anhydrases : CA  In order to character ize and elucidate physiological roles of these CAs, structural kinetic, and biophysical studies have been performed on the CA expressed by the T. cru gammaproteobacterium. CA were shown to be similar to hC A II, which could imply that the T. cru CA could exert similar physiological roles to hCA II. However, significant differences regarding the quaternary structure and the thermal stability were observed. T. cru CA forms a homodimer in
15 solution, which is class CAs. Also, the thermal stability of the T. cru CA was greatly enhanced as compared to hCA II, which could be the result of the presence of a disulfide bond between residues Cys99 and Cys256. The results obtained i n this project are used to provide insights to the function of the T. cru CA in the biological context.
16 CHAPTER 1 INTRODUCTION Carbonic anhydrases (CAs) are mainly zinc metalloenzymes that catalyze the interconversion of carbon dioxide and water to bicarbonate and protons (1 3). Carbonic anhydrase was initially discovered as the enzyme responsible for catalyzing the conversion from bicarbonate to carbon dioxide necessary for rapid transit from the red blood cells to the lung capillaries in mammals (4 ). However, CAs are found in virtually all living organisms and have been shown to be involved in many important and complex physiological processes, such as respiration, pH regulation in tissues, renal tubular acidification, bone resorption, and secretory processes including the formation of cerebrospinal fluid and aqueous humor in animals (3, 5 7). CAs have also been found to play important roles in physiological processes of more primitive organisms, including photosynthesis, CO 2 essential in most living organisms (8, 9). Classification of CAs Three evolutionary and structurally distinct classes of CAs have been established according to sequence similarities and the ov erall folds, both of which indicate different phylogenetic and physiological pathways for each class (10, 11). The first and best CA class, which mostly comprises mammalian CAs. There CA isofor ms isolated from mammals. These vary in CAs are mainly cytosolic (CA I, II, III, VI, XIII), transmembrane or GPI anchored (CA IV, IX, XII, XIV), secretory (CA VI), and mitocho CAs, which up until recently were thought to be exclusively expressed by eukaryotic
17 organisms, have also been found in the periplasmic space of certain prokaryotes, such as the Gram negative bacterium Neisseria gonor rhoeae (12), the nitrogen fixing alphaproteobac terium Mesorhizobium loti (13) and the purple non sulfur bacterium Rhodopseudomonas palustris CAs are monomeric in nature, but there are a few that are dimeric, such as hCA VI, hCA IX CA in R. CA I from green alga Chlamydomonas reinhardtii among others. Most class CAs are roughly spherical enzymes with a highly conserved active site formed by a conical cavity that is approximately 15 deep, as can be observed in hCA II (Figure 1 1) (7). At the bottom of the active site lies the zinc ion tetrahedrally coordinated by 3 conserved histidines and a water molecule/hydroxide ion. The active site cavity is split into two very different environments, where one side of the zinc believed to be where the CO 2 binds is lined with mostly hydrophobic residues, and the other side which leads out of the active site into the bulk solvent is lined with hydrophilic amino acids (Figure 1 2). The dual nature of the active CAs allows the rapid and sustained catalytic conversion of CO 2 to bicarbonate (2). A second, less class CAs, predominantly found in plants, algae, and prokaryotic organisms belonging to b oth CAs was reported in 2000 (17), confirming that the understanding of these enzymes is far behind CAs and the CAs form dimers, tetramers, hexamers, and octamers, suggesting that dimers are the basic building units CAs (18). An example of a crystal
18 str CA from Haemophilus influenzae (PDB ID: 2A8C) is presented in Figure 1 CAs, it can helical content in their secondary structures t han the other two classes of CAs (15). Not much information is available on the CAs are much more class CAs (20). Sequence analyses demonstrated that only 5 CAs, which are the three CAs, the zinc is tetrahedrally coordinated by two cysteines and a histidine plus a fourth ligand, which can be a water molecule CA, denoted Cab, from Methanobacterium thermoautotrophicum (16) an acetate molecule observed CA from Pisum sativum crystallized in acetate (18) or even the conserved CAs, as seen in the Escherichia coli and Porphyridium purpureum CA CAs, the CAs are believed to catalyze the interconversion of CO 2 and bicarbonate through a zinc hydroxide mechanism a s well, explained in detail further below. CA class, initially thought to be solely expressed in methane CA reported was Cam, isolated from Methanosarcina thermophila in 1994 (22) and crysta llized in 1996 (Figure 1 4) (PDB ID: CAs have been crystallized thus far, but they all CAs crystallized to date are homotrimeric structures whose monomers share a distinctive l eft helix fold that is predicted by a unique sequence motif also observed in the superfamily of
19 CA zinc is tetrahedrally coordinated by 3 histidines and a water molecule or hydr oxide ion, but the location of CA is located at the interface between two monomers, where two histidine ligands are donated by one monomer, and the other histidine comes from the ad jacent monomer forming the active site interface (10, 26). Cam was initially thought to be a zinc metalloenzyme, as high level expression in E. coli yields a zinc enzyme. However, substitution of zinc with other transition metals, such as iron and cobalt, yielded higher rates of CO 2 hydration for Cam, implying that native Cam expressed in M. thermophila might function with a different transition metal, with iron appearing to be the physiologically relevant metal (27 29). Independent of the transition metal CAs that are active exhibit a metal hydroxide catalytic mechanism with proton transfer as the rate CAs (10). Catalytic Mechanism of CAs The mechanism underlying the catalytic activit y of CAs has been extensively class CAs share the same overall metal (Zn) hydroxide ping pong mechanism composed of two independent catalytic steps, as shown below in Eq. 1 and 2, where E is the enzyme and M 2+ is a bivalent ion, typically Zn 2+ in CAs (26, 30 32). ( Eq. 1 1 ) ( Eq. 1 2 ) Equation 1 1 shows the first step of catalysis, the hydration of CO 2 to bicarbonate, where a pair of electrons on the metal bound hydroxide (in most CAs, zinc bound
20 hydroxide) nucleophilically attacks the substrate CO 2 to form zinc bound bicarbonate (30, 31). A water molecule subsequently displaces the zinc bound bicarbon ate, leaving a zinc CAs, it has been shown that CO 2 binds in a conserved hydrophobic region of the active site before the nucleophilic attack (2). A more detailed look into the active site of one of the fastest CAs know n, hCA II, shows a hydrogen bond network essential for the hydration of CO 2 formed by residues Thr199 and Glu106. Briefly, hydrogen bonding between the Thr199 hydroxyl and the Glu106 carboxylate oxygen allows the Thr199 hydroxyl group to act as a hydrogen bond acceptor to the zinc bound hydroxide, optimizing the orientation of the electron pair of the hydroxide ion for nucleophilic attack on CO 2 (33). Furthermore, the backbone amide of Thr199 hydrogen bonds with CO 2 sligh tly polarizing the carbon on CO 2 f or nucleophilic attack and providing an environment which promotes the dissociation of bicarbonate for more efficient product removal from the active site (Figure 1 5) (34 36). CAs do not have corresponding residues to Thr199 and Gl u106, but they do have residues that are proposed to have the same catalytic function. CA from Arabidopsis thaliana, and equivalent Gln151 in P. sativum CA, might prov ide the same function as the backbone amide of Thr199 (18, 37, 38). Similarly, the residues CA Cam have been demonstrated to assume the catalytic function of Thr199, where Asn202 is responsible for polarizing the CO 2 molecules and Gln75 primes the hydroxide ion for nucleophilic attack on CO 2 (32). During a sec ond, independent step shown in Eq. 1 2, a proton is transferred from the zinc bound water to the bulk solvent, regenerating the zinc bound hydroxide ion.
21 This proton transfer stage is the rate limiting step of catalysis, which f or hCA II is in the order of 10 6 s 1 (31, 39). In Eq. 1 2, B represents a proton donor/acceptor, which can be a buffer molecule in solution for intermolecular proton transfer, or a proton shuttle residue (PSR) in the enzyme itself. The rate for proton transfer directly from the zinc bound water with a pK a ~7 to the bulk solvent (k cat ) is approximately 10 4 s 1 Enzymes exhibiting a k cat > 10 4 s 1 (reflective of the proton transfer rate) have an intermediate PSR responsible for transferring the proton from the zinc bound water to the bulk solvent (2, 10, 26). In hCA II, a network of highly ordered water molecules at the active site shuttle the proto n to His64, which acts as a PSR, transferring the proton out of the active site to the bulk solvent (40 42). PSRs have also been observed in other CAs, class CA CA Cab from M. thermoautotrophicum (which is CAs), CA Cam from M. thermophila which further underscores the importance of PSRs in the regeneration of the active site in CAs (10, 24, 43, 44). Chemolithoautotrophs and Thiomicrospira crunogena Genomic studies of the novel deep sea vent chemolithoautotroph Thiomicrospira crunogena XCL 2 ( T. cru ) revealed the coding and expression of four different CAs, CA famil ies (45, 46). Chemolithoautotrophs are defined as bacterial organisms that obtain energy from the oxidation of inorganic compounds to fuel carbon fixation using CO 2 as their primary source of carbon (45, 46). Some of these bacteria, including T. cru live in deep sea hydrothermal vents, a harsh habitat where nutrients, such as dissolved inorganic carbon (DIC) and oxygen, are temporally and spatially limited. Although the hydrothermal fluid chemical composition might vary geographically, hydrothermal fluid u sually contains
22 millimolal concentrations of alkali metal cations Li + and K + in solution, as well as alkaline earth cations Ca 2+ Mg 2+ and Sr 2+ which generally form deposits upon reactions with the reduced sulfate anions present in the fluid. Vent fluid also contains high concentrations of some trace minerals, such as manganese, iron, and zinc, as well as high concentrations of reduced sulfur anions, chloride, and low concentrations of bromide (47). The environment at hydrothermal vents is constantly chan ging, as dilute hydrothermal fluid (warm, anoxic, highly reduced, pH 5 8, [DIC] = 2 7 mM) interacts with bottom water (2C, oxic, neutral pH, [DIC] = 20M), leading to oscillatory habitat chemistry dominance (45). Interestingly enough, many of these bacter ia have developed a wide array of adaptations, such as carbon concentrating mechanisms (CCM), symbiotic relationships with invertebrates, and formation of microbial mats or veils to produce a stable microenvironment, which allow them to grow and thrive in these nutrient limited habitats (8). T. cru is a sulfur oxidizing gammaproteobacterium that plays a significant role in the sustainability of deep sea hydrothermal vent communities. It is a spiral shaped, colorless, obligate aerobe isolated from the East P acific Rise deep sea hydrothermal vents that uses hydrogen sulfide and other reduced sulfur species to fuel cell maintenance and carbon fixation (Figure 1 6). T. cru exhibits optimum growth in an environment with a pH range of 5.0 to 8.5, NaCl concentratio n > 45 mM, and temperature range from 4 to 38.5 C (48, 49). T. cru encodes a carbon concentrating mechanism which allows it to rapidly grow under conditions of bicarbonate and CO 2 ds, a critical adaptation to survive in the harsh environment found at the hydrothermal vents (45).
23 Previous results have shown that active transport of bicarbonate or CO 2 plays a role in the CCM. Therefore, the expression of four different CAs suggests a role in bicarbonate and carbon dioxide transport, as well as an indirect role in CO 2 fixation, as part of the CCM (46). Genomic and expression studies have shown that T. cru expresses a CA like protein. Figure 1 7 shows a putative model of the molecular interaction of the CAs as part of the CCM in T. cru CA could involve CO 2 or bicarbonate sequestration to prevent diffusion out of the cell an d facilitate CO 2 diffusion CA expressed in R. palustris ( 14 CA in Helicobacter pylori (50). As CO 2 diffuses into th CA could be associated with maintaining a CO 2 bicarbonate pool near chemical equilibrium in order to facilitate the use of CO 2 by form II ribulose 1,5 bisphosphate carboxylase oxygenase (RuBisCO II), or CO 2 entering the carb CA (CsoCA) is involved in carbon fixation, possibly sequestering CO 2 in the carboxysome in order to be fixated into phosphoglyceric acid (PGA) by RuBisCO I, which has a low affinity for CO 2 and low turnover rates (51). Subsequently, PGA enters the Calvin Benson Bassham cycle and is converted to glyceraldehyde 3 phosphate (G3P), which then enters a reductive tricarboxylic acid cycle (TCA) to form starch, pyruvate, and other carbon skeletons r equired for normal cellular function (45). The T. cru CA To gain insight into the physiological relevance of these CAs in the carbon concentrating mechanism of T. cru CA. The T. cru CA gene encodes a fu ll length protein consisting of 315 amino acid
24 CAs in other previously mentioned prokaryotes prompted the analysis of the amino acid sequence by the SignalP software (52, 53), which predicted a signal peptide N VAAP at the N CA expressed in T. cru contains 292 amino acid residues, with a calculated molecular weight of 33 kDa. Also, its expression in the periplasm of a hydrothermal vent gammaproteobacterium suggests that the structure c ould exhibit features related to increased thermal stability, which could potentially be applied to engineer more thermostable CA isoforms that could be used in industrial applications. Attempts to obtain a T. cru CA knockout mutant have been unsuccessfu l (unpublished data by the Scott laboratory), which suggests that T. cru CA has an essential role in the T. cru We hypothesized that T. cru CA has a similar structure and activity to hCA II, and is possibly i nvolved in CO 2 entrapment and carbon fixation. To complete this project, we performed kinetic studies of the T. cru CA using oxygen 18 ( 18 O) exchange at chemical equilibrium measured by membrane inlet mass spectrometry, which is further explained in C hap ter 2, used differential scanning calorimetry at different pH values to study its thermal stability, and obtained a crystal structure of the T. cru CA by X ray crystallography. The results of these studies are present ed in C hapter 3, and discussed in fur ther detail in C hapter 4.
25 Figure 1 1. C artoon representation of hCA II. The zinc coordinating histidines are represented as sticks, and the zinc ion is shown as a gray sphere. Nitrogen atoms are shown in blue. (PDB ID: 3KS3; 54).
26 Figure 1 2. Cartoon representation of hCA II active site residues (PDB ID: 3KS3; 54). The active site residues are represented as sticks. To highlight the dual nature of the active site, the hydrophilic active site residues are shown in yellow, and the hyd rophobic residues are shown in blue. The zinc ion is represented as a gray sphere. Oxygen atoms are shown in red, and nitrogen atoms in blue.
27 Figure 1 CA expressed in Haemophilus influenzae bacterium (PDB ID: 2A8C; 19) The biological assembly of the protein is a tetramer, and each monomer has its own active site. The zinc ions are shown as blue spheres.
28 Figure 1 4. Cart CA expressed in the thermophile Methanosarcina thermophila (PDB ID: 1QRG; 24). The biological assembly of Cam is a trimer, with each chain presented in a different color (A red, B green, and C cyan). The zinc ion in chain A, shown as a gray sphere, is coordinated by two histidines (His81a, His122 a) in chain A, and one histidine in chain C (His117c). His81b, His122b, His117a, and His81c, His122c, His 117b, coordinate the zinc ion in chains B and C, respectively. Zinc coordinating histidines are represented as sticks, with the oxygen atoms shown in red, and nitrogen atoms shown in blue.
29 Figure 1 5. Hydrogen bonding network involved in the zinc hydroxide mechanism of hCA II. 1) Glu106 hydrogen bonds to the hydroxyl group of Thr199, which acts as a hydrogen bond acceptor to the zinc bound hydr oxide, orientating the electron pair for nucleophilic attack. Also, the backbone amide group of Thr199 polarizes the CO 2 by hydrogen bonding. 2) After nucleophilic attack on CO 2 by the zinc bound hydroxide, the bicarbonate ion is formed and, 3) is removed fro m the active site by displacement of a water molecule. 4) His64 serves as a proton shuttle to transfer the proton from the zinc bound water to the bulk solvent, regenerating the enzyme for catalysis. Modified from Sprigings and Hall, 2001 (55).
30 Fig ure 1 6. Transmission electron micrograph of Thiomicrospira crunogena XCL 2 cells. The T. cru cells are colorless, spiral shaped, gram negative bacteria (0.4 0.5 x 1.5 3.0 m 2 ) that contain a single flagellum (not shown in figure). Provided by Dr. Kath leen Scott.
31 Figure 1 7. Putative model for the interaction of four carbonic anhydrases in the T. cru CA (red) could sequester CO 2 or bicarbonate to prevent diffusion out of the cell. As CO 2 diffuses into the CA (blue) could maintain an equilibrium of the CO 2 /bicarbonate pool, until CO 2 is used by form II ribulose 1,5 bisphosphate carboxylase oxygenase (RuBisCO II) or CA; yellow) is involved in carbon fixation, probably by sequestering CO 2 in the carboxysome in order to be fixated into phosphoglyceric acid (PGA) by RuBisCO I, which has a low affinity for CO 2 and low turnover rates (51). As part of the carbon fixation, PGA continues on the Calvin Benson Bassham cycle and is converted to glyceraldehyde 3 phosphate (G3P), which then enters a reductive tricarboxylic acid cycle (TCA) to form starch, pyruvate, and other car bon skeletons required for normal cell function (45).
32 CHAPTER 2 MATERIALS AND METHOD S Expression and Purification of T. cru nogena CA A pET SUMO plasmid containing the T. cru CA gene was transformed into BL21(DE3) E. coli cells through standard protocols for high yield protein expression, and the transformed cells were incubated at 37C in a modified Luria Broth with 2x yeast extract and tryptone (2XYT broth) containing 50 g/mL kanamycin at 200 rpm to an OD 600 of 0.6 1. 0 AU (46, 56) T. cru CA expression was induced for 18 h at 18C D thiogalactopyranoside (IPTG) to a final concentration of 0.4 mM. The cells were harvested at 4C by centrifugation at 4000 rpm for 10 min and were kept overnig ht at 20 C The cell pellets were resuspended in lysis buffer (0.1 M Tris HCl pH 9.0, 0.2 M Na 2 SO 4 ) with 1 mg/mL lysozyme and 50 g/mL DNAse I, and were lysed at 4C stirring overnight. The samples were then centrifuged for 70 min at 15000 rpm. T. cru CA was purified through affinity chromatography using a p ( amino methyl) benzenesulfonamide (p AMBS) resin as has been previously described for hCA II purification (57), with several adjustments. Briefly, the cell lysate was loaded onto the affinity column equilibrated with 10 column volumes (CV) of the lysis buffer. The column was washed with at least 20 CV of the lysis buffer and 20 CV of the lysis buffer at pH 7.0, until the absorbance of the flow through measured at 280 nm was below the detection limit f or proteins, 0.1 AU, after each wash. The protein was eluted with a sodium azide buffer (50 mM Tris HCl pH 7.8, 0.4 M sodium azide), after which the eluted samples were buffer exchanged and concentrated using an Amicon Ultra 15 Centrifugal Filter Unit to r emove the sodium azide. Further purification of the protein
33 required a second step of p AMBS affinity chromatography. The protein samples were desalted and concentrated using the Amicon centrifugal filter. The final sample concentration was determined by UV Vis spectroscopy at 280 nm, using an extinction coefficient of 42985 M 1 cm 1 calculated from the amino acid sequence (58), followed by SDS PAGE analysis to assess purity (59). Gel Filtration Chromatography A 30 M sample of T. cru CA was analyzed on a GE Healthcare KTA Fast Protein Liquid Chromatography (FPLC) protein purification system, equipped with a prepacked HiPrep 16/60 Sephacryl S 200 High Resolution gel filtration column (Exclusion range: 5 to 250 kDa; GE Healthcare Biosciences AB, Sweden ) previously equilibrated with 50 mM Tris HCl pH 7.0, 100 mM NaCl buffer. Data acquisition and processing were performed using the FPLC UNICORN software. The protein was eluted with the aforementioned buffer at a 0.1 mL/min flow rate, collecting 1 mL fract ions. Protein fractions were detected by absorbance at 280 nm, and stored at 4C. The Sephacryl S 200 column was calibrated by using Gel Filtration Standard molecular weight markers from Bio Rad (Cat. no. 151 1901), following the provided instructions. Cry stallization and X ray Data Collection of T. cru CA Initial crystallization screening was performed in INTELLI PLATE 96 well sitting drop vapor diffusion crystallization plates (Art Robbins Instruments, Sunnyvale, California, USA) using a Crystal Gryphon Protein Crystallization System (Art Robbins Instruments, Sunnyvale, California, USA) with four commercial screens: Crystal Screen, Crystal Screen 2, PEG/Ion Screen, and PEG/Ion2 Screen (Hampton Research, Aliso Viejo, California, USA). Drops consisting of ~0.3 mM protein sample (in 50 mM Tris HCl pH 7.8, 100 mM NaCl) and precipitant solution at two different ratios (1:1 and 2:3
34 protein/precipitant solution) were equilibrated at 17C against a reservoir containing 100 L of the precipitant solution. Small pr otein crystals were obtained in 2% v/v Tacsimate pH 4.0, 0.1 M Sodium acetate trihydrate pH 4.6, 16% w/v PEG 3350 (Solution 31 from PEG/Ion2 Hampton Screen). The crystals were cryoprotected by rapidly immersing it into the precipitant solution with 20% gly cerol prior to freezing in liquid N 2 X ray diffraction data was collected using an ADSC Quantum 270 CCD detector at The data sets were collected at a crystal to detector dis tance of 300 mm, 1 oscillation angle with an exposure time of 60s per image for 180 frames. The data were indexed, integrated and scaled to a 2.6 resolution with HKL2000 software (60). Structure Determination of T. cru CA The crystal structure of hCA II (PDB ID: 3KS3; 54) was used to calculate the initial phases of the T. cru CA using the PHENIX AutoMR software (61, 62). The zinc and all solvent molecules were removed to decrease model bias. PHENIX AutoMR yielded a coordinate file with 4 molecules in the asymmetric unit, and two Fourier electron density map files, a 2F o F c map which shows density observed from the new model and a F o F c map which shows the difference between the observed density in the model and experimental data. The model obtained fr om AutoMR was truncated, where amino acid residues that had no density were removed, and additional residues were built into observed density. The truncated model was used for a second round of molecular replacement with PHENIX AutoMR. The coordinate file and electron density maps obtained from the second round of molecular replacement were used to build in the residues that were missing in the truncated model to complete the structure of T. cru CA. Structure refinement was completed by alternating the us e of the PHENIX software
35 package with density modification using AutoB uild (63) to enhance the Fourier electron density maps and manual refitting of the model in Coot ( 64). 5% of the unique reflections were selected randomly before initiating refinement and were excluded from refinement for purposes of R free calculation (65). The validity of the model was assessed by PROCHECK (66). Secondary structure moieties were assig ned using the S TRIDE program (67). Figures were made using PyMOL. Oxygen 18 Exchange Kinetic Analysis The 18 O exchange experiments presented in this thesis were conducted by Dr. Chingkuang Tu in the Silverman laboratory. The method is based on the measu rement of the depletion of 18 O from species of CO 2 at chemical equilibrium by membrane inlet mass spectrometry (68, 69). A continuous measure of various isotopic species of CO 2 is provided by CO 2 diffusing across a membrane permeable to dissolved gases, wh ich is submerged in the reaction solution and connected by glass tubing to a mass spectrometer (Extrel EXM 200) (70). The catalyzed and uncatalyzed exchange of 18 O between CO 2 and water at chemical equilibrium were measured in the absence of added buffer a t a total substrate concentration of 25 mM. The reaction is kept without added buffers to simplify data analysis, since buffers can interfere with proton transfer. The reaction solution was maintained at 25C, and the ionic strength of the solution was kep t at 0.2 M by addition of Na 2 SO 4 During the first independent stage of catalysis, there is a probability that dehydration of the labeled HCO 3 will label the active site with 18 O (Eq. 2 1). During the second catalytic stage, the zinc bound 18 O labeled hy droxide is protonated, forming H 2 18 O, which then diffuses into the solvent (Eq. 2 2). (Eq. 2 1)
36 (Eq. 2 2) The 18 O exchange kinetic approach to measure the catalysis of CA yields two rates. The first, denoted R 1 describes the rate of intercon version of carbon dioxide and bicarbonate at chemical equilibrium (Eq. 2 3). k cat ex is a rate constant for maximal interconversion of substrate to product, in this case CO 2 and HCO 3 whereas K eff CO2 is an apparent affinity constant for substrate to the en zyme, either CO 2 or HCO 3 to carbonic anhydrase. The ratio k cat ex /K eff CO2 is equivalent to the catalytic efficiency (k cat /K M ) of hydration obtained by steady state methods (71). (Eq. 2 3) The second rate obtained by the 18 O exchange method, R H2O describes the rate at which 18 O labeled water is released from the active site. R H2O is dependent on donation of protons to the 18 O labeled zinc bound hydroxide by His64 as a second independent step of the catalysis by hCA II, or its equivalent in other isozymes, as shown in Eq. 2 2 (68, 72). In Eq. 2 4, k B is the rate constant for proton transfer to the zinc bound hydroxide, and (K a )His64 and (K a )Zn H2O are the ionization constants of the proton donor His64 and the zinc bound water molecule, respectively. (Eq. 2 4) Equation 2 3 and 2 4 were fitted to the data by using non linear least squares methods in Enzfitter (Biosoft).
37 Differential Scanning Calorimetry (DSC) Differential scanning calorimetry (DSC) experiments were performed to assess the therm ostability of T. cru CA using a VP DSC microcalorimeter (Microcal, Inc., North Hampton, MA) with a cell volume of ~ 0.6 mL. T. cru CA samples were buffer exchanged into solutions with pH values ranging from 4 to 9, at 1 pH unit intervals. A phosphate/c itrate buffer was used for samples at pH values 4 6, and a 50 mM Tris HCl, 100 mM NaCL buffer was used for samples at pH values 7 9, all of which had a protein concentration of 30 M. The protein and buffer samples were degassed, while stirring, at 16C fo r 20 minutes prior to data collection. DSC scans were collected from 30C to 90C with a scan rate of 60C/hr. DSC scans for all the samples were performed in triplicate. A reference scan for each pH value, where buffer was placed in both the reference and sample cells, was also performed in order to subtract the contributing heat capacity of the buffer solution. After subtracting the reference and adjusting the baseline for each scan, the calorimetric enthalpies of unfolding were calculated by integrating the area under the peaks in the thermograms. The thermograms were fit to a non two v ) (73). The melting temperature (T M ) values of the T. cru CA samples at the different pH values and of hCA II were obtained from the midpoints of the thermograms.
38 CHAPTER 3 RESULTS CA expressed in Thiomicrospira crunogena was successfully overexpressed and isolated with a usual yield of 10 mg per liter of bacterial culture. Small, rectangular crystals with approximate dimensions 0.5 x 0.5 x 0.01 mm 3 formed after 14 days in a variety of crystallization conditions containing PEG 3350 over a pH range of 4. 0 7.4 (Figure 3 1). Crystals grown by hanging drop vapour diffusion method in 2% v/v tacsimate pH 4.0, 0.1 M sodium acetate trihydrate pH 4.6, 16% w/v PEG3350 and at CHESS synchrotron. Diffraction data at 2.6 resolution was collected at CHESS synchrotron. The crystals were shown to belong to the C2 space group, with unit cell parameters a = 127.1, b = 102.2, c = 105.0 = 127.3 with an R sym of 10.0%. Initial data processing with HKL2000 software (60) also provided the possibility of an F222 space group, with unit cell parameters a = 102.2, b = 127.0, c = 167.2 but with an R sym of ~ 50%, which led to the assignment of C2 as the space grou p. Data collection statistics and processing parameters are summarized in Table 3 1. Considering the C2 space group, the unit cell parameters, and assuming the protein molecular weight was 33217 Da with 4 molecules in the crystallographic asymmetric unit, a Matthews coefficient (V M ) of 2.04 3 Da 1 was calculated, with an estimated solvent content of 39.75% (74, 75). The diffraction data were phased using the molecular replacement method with the structure of hCA II (PDB ID: 3KS3; 54) using PHENIX AutoMR. This yielded a unique solution, comprising of 4 molecules in the asymmetric unit with a final translation function Z score (TFZ) of 17.9. A TFZ score above 8 usually indicates that the MR was
39 successful. The structure was then refined using standard proto cols to 2.6 resolution with a R work of 20.8% and R free of 24.4%. The average B factor for the zinc molecules was 21.6 2 The overall B factor for the main chain and side chai n atoms were 30.3 2 and 33.3 2 respectively. A total of 104 solvent molecule s were added, with an average B factor o f 28.5 2 Root mean square deviation (RMSD) values from ideal bond lengths and angles were 0.003 and 0.9, respectively (Table 3 1). The structure of T. cru CA contains 4 molecules in the asymmetric unit, which appear to be interacting as two separate dimers (Figure 3 2). Previous assays used to analyze the protein samples were performed under denaturing conditions and provided structure was the first data to suggest the possibility of a dimer. Size exclusion chromatography using a Sephacryl S 200 FPLC column, along with DSC studies performed later, strongly supported that the protein is dimeric in solution. The crystal structur e of T. cru CA demonstrates the possibility of an interface between the two dimers, but there is no supportive evidence to suggest that the protein acts as a dimer of dimers. Hence, it is believed that the basic unit of the enzyme is a dimer, and the app earance of a dimer of dimers in the MR solution is a result of crystal packing. Residues 75 304 are observed for all chains in the structure, except for Chain B, which contains three additional residues (Arg305, Asn306, and Ala307, which is supposed to be arginine, but it had no side chain density) at the C terminus (Figure 3 2). No residues before Pro75 were observed for any of the chains, even after multiple rounds of refinement, which in addition with other computational data suggests that the N termi nus might be disordered (details in discussion).
40 T. cru CA was superimposed onto hCA II (PDB ID: 3KS3) using secondary structure matching in Coot with a RMSD of 1.455 Comparison of the T. cru CA monomer with hCA II shows minor differences in the ov erall structure, mostly in the strands, and turns (Figure 3 3), but there is a definite structural similarity between both enzymes. Each subunit counts with a zinc molecule tetrahedrally coordinated by His165, His167, His 184 and a water molecule, as is CAs. The active site of the T. cru CA, shown larger in Figure 3 4, superimposes well over the hCA II active site and most of the active site residues are conserved, except for Ala65 and Asn67, which are Thr141 and Gln143 in T. cru CA, respectively. The catalytic activity of the T. cru CA was also assayed and compared to that of hCA II. As mentioned in the Methods section, measurement of the catalytic activity by 18 O exchange yields two rates. The first rate obtained is R 1 (Eq. 2 3), from which we determined k cat ex /K eff CO2 equivalent to the catalytic efficiency, for the forward direction as it is expressed in Eq. 2 1. The pH profile for this rate constant was fitted to a single ionization event, g iving a maximal k cat /K M of 11 1 M 1 s 1 and an estimated pK a for the zinc bound water of 6.6 0.1. The values obtained for hCA II and T. cru CA kinetic assays are listed in Table 3 2. The pH profile of T. cru CA for R 1 appeared qualitatively similar to the pH profile of hCA II for R 1 as both had a bell shaped curve with the maximal R 1 near neutral pH, though it can be appreciated that the pH profile of the T. cru CA is shifted towards the left, obtaining a maximal R 1 at a lower pH than hCA II (Figure 3 5). The pH profile of the catalytic efficiency (k cat /K M ) for both enzymes was also remarkably similar, with a maximal catalytic efficiency at high pH,
41 corresponding to the reactivity of the zinc bound hydroxide in the h ydration reaction (Figure 3 6). However, there were significant quantitative differences between the kinetic parameters for the T. cru CA and hCA II. The catalytic efficiency of T. cru CA, as measured by k cat ex /K eff CO2 was reduced 10 fold as compared to hCA II, and the kinetic pK a which is an estimated value of the pK a of the zinc bound water, determined from the pH profile of k cat ex /K eff CO2 was lower by approximately one unit (Table 3 2). The second rate obtained from the 18 O exchange kinetic assays i s R H2O (Eq. 2 4), the rate of release of H 2 18 O from the active site, which is dependent on proton transfer to the labeled zinc bound hydroxide. Intramolecular proton transfer determines the values for R H2O /[E], as has been demonstrated by pH profiles, kine tic isotopic effects, and chemical rescue experiments (69, 76). The pH profiles for R H2O /[E] have a characteristic bell shaped curve for most of the pH range covered in the studies (Figure 3 7), which is attributed to the transfer of a proton from the PSR His64 to the zinc bound hydroxide (Eq. 2 2). The solid line in Figure 3 7 represents the fit of Eq. 2 4 to the data. A second, dashed line that appears to fit the data more accurately is the result of a double ionization model used to calculate the kinetic parameters for R H2O /[E]. In order to fit Eq. 2 4 to the data accurately, the pK a values for the donor and acceptor have to be assigned, which meant using the pK a value for the zinc bound water as determined by 18 O exchange for k cat ex /K eff CO2 in the hydrat ion direction. The fit of Eq. 2 4 to the data yielded a rate constant for intramolecular proton transfer (kB) of 0.30 0.05 s 1 The rate constant k B was more similar between T. cru CA and hCA II than k cat /K M (Table 3 2), suggesting that T. cru CA has a PSR similar to the side chain of His64 in hCA II for proton tr ansfer. As the pH profile for R 1 the pH profile of T. cru CA for R H2O /[E] was
42 also shifted toward a lower pH (Figure 3 7). The pK a of the zinc bound water and the proton shuttle r esidue in T. cru CA are both 6.4 0.1, which could be a reason why proton transfer is slower than in hCA II. T. cru CA activity was also assayed in the presence of various anions that can be found in the hydrothermal vents to determine whether the an ions in the environment modulate its catalytic activity. Inhibition constants of iodide, chloride, and bromide were obtained for T. cru CA using the 18 O exchange method (Figure 3 8) and compared to the inhibition constants for hCA II reported previously (Table 3 3) (77), demonstrating similar inhibition values for both enzymes. Inhibition by hydrogen sulfide (HS ), one of the major sulfur species available at hydrothermal vents, was also obtained for T. cru CA and hCA II (Table 3 3). Interestingly, resu lts show that HS actively inhibits both T. cru CA and hCA II with micromolar affinity, which somewhat conflicts with the sulfur oxidizing nature of the T. cru gammaproteobacterium. The thermal stability of T. cru CA was determined at different pH val ues ranging from 4 to 9 by DSC, and compared to the thermal stability of hCA II at pH 8. A preliminary temperature profile of T. cru CA was determined using 18 O exchange kinetics by increasing the temperature during the assay, which showed that the therm al inactivation temperature was between 55 and 60C (data not shown). The melting temperature (T M ) of hCA II, also the thermal inactivation temperature, is 59.5 0.5 C, and is observed as a single endothermic peak at the midpoint of the DSC curves repres entative of the main unfolding transition (78). Unlike the scans collected for hCA II, the thermograms collected for the T. cru CA samples presented two independent transitions, one at approximately 59C and the other at 72C, for all pH values at which
43 data were collected (Table 3 4). We attempted to collect data for T. cru CA at pH 4 in several occasions, keeping sample concentrations roughly the same, but we were unsuccessful, which led us to believe that the enzyme is unstable at pH 4. It was also d etermined by completing a reverse scan that only the first transition was reversible, suggesting that the first transition represents the dissociation of the dimer while the second transition represents the unfolding of the T. cru CA. The thermograms wer e initially fit to a two v ), but the model did not fit the DSC data accurately (73). This led us to exchange the two state reversible model for a non two state reversible unfolding model, indicative of an unfolding intermediate. After fitting the data to a non 2 v ), and the melting temperatures were calculated and are listed in Tab le 3 4. The melting temperatures of the first and second transition were plotted as a function of pH (Figures 3 9 and 3 10) to determine if the changes in environmental pH affected the thermal stability of the T. cru CA. The T M for the lower temperature transition increased roughly 1 2C per increase in pH unit, while the T M for the second transition increased approximately 3 throughout the pH profile. A linear relationship could be established between pH and temperature of the first transition, with a correlation coefficient (r) of 0.94. A linear fit was also applied to the second transition, but the r value is much lower temperature for the second transition. However, the pH profile for the melting temperatures for both transitions, more so for the first transition, demonstrated that
44 there is a direct relationship between pH and temperature for T. cru CA, as the thermal stability of the enzyme was enhanced as pH was i ncreased.
45 Table 3 1. Data collection and refinement statistics for the crystallographic study of T. cru CA. Data collection statistics Space group C2 Unit cell parameters (,) a = 127.1, b = 102.2, c = 105.0 Resolution range () 20.00 2.60 (2.69 2.60)* R sym (%) a 10.0 (40.5) 9.2 (2.5) Redundancy 3.7 (3.5) Total number of measured reflections 120808 Total number of unique reflections 32859 R work (%) b 20.8 R free (%) c 24.5 V M ( 3 Da 1 ) 2.04 Residue Nos. Chain A: 75 304 Chain B: 75 307 Chain C: 75 304 Chain D: 75 304 No. of Atoms Protein Zn H 2 O molecules 7537 4 104 B factors ( 2 ), average Main chain, Side chain, Zn Solvent Chain A: 28.5, 31.2,19.6 Chain B: 28.1, 31.2,18.0 Chain C: 32.2, 35.2, 22.7 Chain D: 32.2, 35.5, 26.3 28.5 Ramachandran statistics (%) Most favored, Additionally allowed, Generously allowed Chain A: 91.8, 6.2, 2.1 Chain B: 93.4, 5.6, 1.0 Chain C: 93.3, 6.2, 0.5 Chain D: 93.8, 5.7, 0.5 R.M.S.D. for bond lengths and angles (,) 0.003, 0.9 Values in parentheses refer to the highest resolution shell. a R sym hkl ) x 100, where I hkl is the intensity of an individual reflection and is the average intensity for this reflection. b R work obs | |F calc obs |] x 100 c R free is calculated the same as R cryst except it uses 5% of the reflection data omitted from refinement.
46 Table 3 2. Comparison of maximal (pH independent) catalytic parameters for T cru CA and hCA II. Parameter T. cru CA hCA II a k cat ex /K eff CO2 (M 1 s 1 ) 11.0 0.1 120 k B (s 1 ) 0.30 0.05 0.8 pK a ZnH2O 1 6.6 0.1 6.9 pK a ZnH2O 2 6.4 0.2 6.8 pK a PSR 2 6.4 0.2 7.2 a (39). Standard errors are no larger than 20%. 1 Determined by 18 O exchange from calculation of k cat ex /K eff CO2 in the hydration of CO 2 2 Determined from calculation of R H20 /[E].
47 Table 3 3. Comparison of inhibition constants for T. cru CA and hCA II. Inhibition constant T. cru CA hCA II a K I (Cl ) 361 27 200 K I (I ) 53 3 26 K I (Br ) 242 55 K I (HS ) 0.0011 0.0001 0.0028 0.0002 a (77)
48 Table 3 4. Thermodynamic parameters of unfolding of T. cru CA at different pH values. pH First transition Second transition T M (C) (kcal/mol) v (kcal/mol) T M (C) (kcal/mol) v (kcal/mol) 5 57.7 0.7 130 33 91 17 68.1 0.8 193 34 75 12 6 58.3 0.4 85 15 123 20 69.5 0.4 234 18 69 7 7 59.8 0.5 211 30 104 15 69.9 0.6 189 30 102 23 8 60.1 0.3 129 12 109 11 71.9 0.4 128 13 103 13 9 62.1 0.4 242 25 98 11 71.0 0.5 119 24 132 28 Control (hCA II, pH 8.0) 55.95 0.05 170 4 181 5
49 Figure 3 1. Crystals of T. cru CA grown in 2% v/v tacsimate pH 4.0, 0.1 M sodium acetate trihydrate pH 4.6, 16% w/v PEG 3350 at 17C using the hanging drop vapour diffusion method. The crystal dimensions are approximately 0.05 x 0.05 x 0.01 mm 3
50 Figure 3 2. Crystal structure of T. cru CA. The crystal contained four molecules in the asymmetric unit (au). Cartoon representation of T. cru CA, with each chain within the au presented in a different color (A purple, B yellow, C green, and D blue). Chains A and B interact to form a dimer, as well as chains C and D. The zinc ions are presented as gray spheres.
51 Figure 3 3. Superposition of the T. cru CA (green) monomer and the hCA II (gray) crystal structure (PDB ID: 3KS3; 54). The cartoon representation overlaps to show the similarities and differences between both structures, with an RMSD of 1.455 The coordinating histidines and the disulfide bond are presented as sticks. The nitrogen is shown in navy blue, sulfur in y ellow, and the zinc ion is shown as a gray sphere.
52 Figure 3 4. Close up stick representation of superimposed active site residues in T. cru CA (green) and hCA II (gray) (PDB ID: 3KS3; 54). Oxygen atoms are shown in red, nitrogen atoms in navy blue and the zinc ion is represented as a gray sphere.
53 Figure 3 5. The pH profiles for R 1 for the hydration of CO 2 catalyzed by T. cru CA (red, ) and hCA II (black, ). Data were collected by 18 O exchange by CO 2 and water measured at 25 C. Total concen tration of all species of CO 2 was 25 mM and sodium sulfate was added to maintain ionic strength at 0.2 M. The solid lines are a fit of Eq. 2 3 to the data. 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06 5 6 7 8 9 R1/[E] pH
54 Figure 3 6 The pH profiles for k cat exch /K eff CO2 (M 1 s 1 ) for the hydration of CO 2 catalyzed by T. cru CA (red, ) and hCA II (black, ). The same experimental conditions were used as in Figure 3 5. The solid lines are a fit of a single ionization model to the data. 1.E+05 1.E+06 1.E+07 1.E+08 1.E+09 5 6 7 8 9 kcat/Km pH
55 Figure 3 7. The pH profile for R H2O /[E] (s 1 ) for proton transfer in the dehyd ration direction catalyzed by T. cru CA (red, ) and hCA II (black, ). The same experimental conditions as in Figure 3 5 were used. The solid lines are a fit of Eq. 2 4 to the data, and the dashed lines are a fit of a double ionization model to the data 1.E+03 1.E+04 1.E+05 1.E+06 5 6 7 8 9 RH2O/[E] pH
56 Figure 3 8. The inhibition of T. cru CA by iodide (black, ), chloride (blue, ), and bromide (red, ). Total concentration of all species of CO 2 was 25 mM in a solution containing 100 mM HEPES at pH 7.6 and 25 C. Na 2 SO 4 was added to maintain ionic strength at a minimum of 0.2 M. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 0.5 1 1.5 Relative Activity [Inhibitor] M
57 Figure 3 9. The pH profile of the melting temperature (TM) of the dissociation of dimeric T. cru CA. DSC thermograms were collected at pH range 5 9 for T. cru CA in triplicate. Af ter data processing, the TM were averaged and plotted as a function of pH. The solid line is a linear fit to the data with a correlation coefficient (r) of 0.94. Error bars are shown. 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 Temperature ( C) pH
58 Figure 3 10: The pH profile of the melting temperature (TM) for the unfolding of dimeric T. cru CA. DSC thermograms were collected at pH range 5 9 for T. cru CA in triplicate. After data processing, the TM were averaged and plotted as a function of pH. The solid line is a linear fit to the data with a correlation coe fficient (r) of 0.80 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 5 6 7 8 9 Temperature () pH
59 CHAPTER 4 DISCUSSION class CA expressed in the T. cru gammaproteobacterium by structural, kinetic, and biophysical means to understand its physiological role. Measurement of 18 O exchange at chemical equilibrium by membrane inlet mass spectrometry was used to obtain the kinetic rates of catalytic activity for T. cru CA, which demonstrated that it is a fast enzyme, and differential scanning calorimetry was used to determine the increased thermal stability of the T. cru CA as compared to hCA II. X ray crystallography allowed us to determine the molecular str ucture for the T. cru CA, which provided structural evidence supporting its fast activity and thermal stability, and revealed the possible dimeric nature of the protein. The interpretation and combination of the results obtained with these techniques, al ong with the comparison to various well CA isoforms, particularly hCA II, has led us to establish a structure function relationship of the T. cru CA that can provide further insight to its physiological role in T. cru Crystal Structure of T. c ru CA The crystal structure obtained for the T. cru CA contained four molecules in the asymmetric unit, with 16 molecules total per unit cell (Figure 4 1). The different chains observed were labeled A, B, C, and D. Each chain contains 230 amino acids, from residue 75 to 304 of the full length sequence, e xcept for chain B, which contains 3 additional residues at the C terminus. Superimposition of chains B, C, and D to chain A, which we selected as the reference chain, showed an RMSD for the superimposition of the corresponding C atoms (residues 75 to 304) of 0.229, 0.228, and 0.243 respectively. Given that the molecules show no significant structural differences, any
60 discussion regarding the monomeric unit of T. cru CA can be applied to all chains in the asymmetric unit. The electron density of some polar residues at the surface of the molecules is weak or unobserved, but the density for the main chain is clearly observed in the final 2Fo Fc map and the density modified map created using AutoBuild. There were significant peaks in the Fourier differ ence map at the active site, indicating the presence of the zinc ion and the zinc bound water molecule. The model contains 104 water molecules, with an average B factor of 28.4 2 Due to the resolution of the data, only a few water molecules could be add ed with absolute certainty, resulting in a reduced number of solvent molecules. This affects our ability to make observations regarding the water network in T. cru CA, which potentially be used to explain differences in the catalytic activity in comparis on to hCA II. The geometry of the final model was verified using PROCHECK (66). The RMSD values from the ideal bond lengths and angles were within the acceptable limits. The ramachandran plot generated by PROCHECK (Figure 4 2) shows that over 90% of the d ihedral angles were within the most favored region, while the rest were in the allowed region, except for 1.0% which were in the generously allowed region. Furthermore, the average B factors of the main chain and side chain atoms are 30.3 2 and 33.3 2 r espectively, with an average B factor for the zinc atom at 21.6 2 suggesting that the structure is correctly built. The T. cru CA monomer has a roughly ellipsoidal shape of approximately 44 x 40 x 41 3 in size. As can be observed in Figure 4 3, the c ore of the molecule is formed by a ten CAs. The active site of
61 the protein is located in a large conical cavity extending from the surface of the molecule to the center, with the zinc ion tetrahedra lly coordinated by His165, His167 and His184, along with a water molecule, found at the base of the cavity (Figure 4 4). The active site is partitioned into two different environments, a hydrophobic patch formed by residues Val186, Val196, Leu251, Val260, and Trp262, corresponding to the hydrophobic patch in hCA II, and an area lined by hydrophilic residues Tyr80, Asn138, His140, Thr141, Gln143, Thr252, and Thr253. Two residues that are part of the hydrophilic patch in hCA II, Ala65 and Asn67, are not conse rved in the active site of T. cru CA; instead, they are replaced by polar residues threonine (Thr141) and glutamine (Gln143) respectively, conserving the hydrophilic nature. Therefore, the same class CAs active site i s maintained in the T. cru CA. Furthermore, the zinc which hydrogen bonds to the amide nitrogen of Thr199 in hCA II (79), are observed in the active site of T. cru CA. Superimposition of T. cru CA with hCA II highlights the structural differences between both enzymes (Figure 3 3), which include changes in secondary structure elements, differences in surface loop lengths, and displacement of certain loops and residues. Residues pertaining to hCA I I are noted in parenthesis. One of the first differences noted is the difference in secondary structural elements. As mentioned CA fold formed by the ten sheet is observed in T. cru CA and superimposes well over strands present in the structure. However, there are some variations regarding the strands, and other strands in hCA II are not
62 identified as such by the secondary struc ture assignment program STRIDE (67). For strand formed by residues Lys(39) and Tyr(40) in hCA II, which is part of strands that form the core, is present as a loop in T. cru strand formed by residues Phe177 Asn1 78 found in T. cru CA is classified as a loop strand formed by residues Ser(173) Asp(175) in hCA II is absent in T. cru strand not observed in hCA II containing residues Ile226 Lys231 appears next to wh strand is in hCA II. Furthermore, strands that are conserved between both proteins appear to be displaced in the strand formed by residues Phe124 Tyr127 is displaced 2.4 strand in hCA II formed by residues Leu(47) Ser(50). strand containing residues Tyr151 strand in hCA II, with residues Val(78) Leu(81). However, none of the displacements strands disrupt the orientation or positi on of the active site residues. The helical content of T. cru sheet when compared to hCA II. The primary differences in the helical regions are the missing helices in T. cru CA. A helix contain ing residues Thr(125) Asp(139) in hCA II is not observed in T. cru CA, and instead is replaced by a shorter, hairpin loop containing residues Asp189 Gly192. Some residues in this helix have been implied in stabilizing the strain observed in the loop containing the cis Pro(202) and catalytic residue Thr(199) in hCA II (80). Loss of this particular helix could result in a decrease of conformational stability. However, T. cru CA contains a disulfide bridge that stabilizes the Thr(199) loop. The disulfi de bond will be discussed later in further detail. A shorter helix, composed of residues Pro(181) Leu (184), is also missing from the T. cru CA
63 helices and variations strands. Most notably, the helix formed by residues Val273 Leu283 is displaced 2.3 from the corresponding helix consisting of residues Ser(220) Arg(227) in hCA II. The largest variability observed between the structures o f T. cru CA and hCA II is due to the difference of surface loops, particularly loops that are shorter or absent in T. cru CA. In T. cru CA, there is only one small, hairpin loop with three residues (Ala93, Pro94, Glu95) added to the N terminal segmen t in T. cru CA, joining two short helices, but missing in hCA II, which instead has the two helices connected by residues Phe20 and Pro21. However, most of the differences in surface loops are observed as deletions in T. cru CA. The major deletions in T. cru CA are the surface loops formed by residues Gly(98) Gln(103) and Lys(228) Met(241), which are extended from behind the active site out to the surface and near the opening of the active site cavity, respectively. The loop Gly(98) Gln(103) is close to the helix formed by Ser(220) Arg(227). Deletion of the loop could account for the displacement of the helix Val273 Leu283, which corresponds to the helix Ser(220) Arg(227) in hCA II, as the helix shifted 2.3 toward the space occupied by th e loop present only in hCA II. Deletion of the surface loop containing residues Lys(228) Met(241), which is located near the opening of the active site, could also account for the displacement of helix Val273 Leu283. Also, all the surface loop deletion s, along with the deletion of helices Thr(125) Asp(139) and Pro(181) Leu(184), result in the appearance of a more CA with a more solvent accessible active site (Figure 4 5).
64 The N terminus of T. cru CA The gene for T. cru CA encodes a full length sequence of 315 amino acids. However, out of the 315 residues encoded, only 292 amino acid residues should be observed. T. cru CA was identified as a periplasmic protein, as its sequence contains the signal peptide NVAAP at th e N terminus, identified by Signal P software, and its periplasmic location was previously confirmed (46, 52, 53). However, even after cleavage of the signal peptide, 64 residues are still unaccounted for in the crystal structure, 52 of which are at the N terminus and the rest located at the C terminus. Before we were able to crystallize the protein and obtain the 3 D structure, we used sequence alignments and homology modeling with hCA II to have a model to work with and establish our hypotheses regarding catalysis and thermal stability (Figure 4 6). In the homology model we obtained, the 52 amino acids at the N terminus were not taken into consideration to build the model; the N terminus was missing. We used the NCBI Protein Blast to search the databases f or possible similar regions between all available protein sequences, including those without reported 3 D structures, and the 52 missing residues at the N terminus of T. cru CA. We found that the sequence (MAAPLIDLGAEAKKQAQKSAATQSAVPEKESATKVAEKQKEPEEKAKP EPKK) is unique among known protein sequences to date. The N terminal region appears to be of hydrophilic nature, as it contains 20 charged residues, among them 11 lysines, 8 glutamates, and one aspartic acid, and 9 polar residues, which are four glutamine s, three serines, and two threonines. The 11 lysines could confer a more basic, rather than acidic nature to the N terminus, which could potentially be the reason why we observe a lower pK a for the zinc bound water in T. cru CA. The lower catalytic effi ciency of T. cru
65 CA that we observed as the result of the kinetic analysis may therefore be a result of decreased nucleophilicity of the zinc bound hydroxide due to the more acidic pK a After obtaining the crystal structure, and noticing that the 52 resi dues at the N terminus were missing, we hypothesized that the missing segment could be intrinsically disordered. Intrinsically disordered regions in proteins have been associated with biological functions regarding signaling, molecular recognition, and reg ulation (81, 82). We used the meta predictor for intrinsically disordered residues PONDR FIT to predict the disordered regions of T. cru CA (81). Figure 4 7 demonstrates that some residues of the missing N terminal region (Ser46 Val48, and Ala54 Glu6 3), and the residues 305 315 at the C terminus missing from the crystal structure, cross the dashed lines at 0.5 of the Y axis, and are therefore predicted as disordered by PONDR FIT. Therefore, although the region is not completely disordered, the various stretches that are disordered within the N terminus could increase the flexibility of that region, leading to the absence in the crystal structure. The Disulfide Bond The monomers in the T. cru CA structure show an intramolecular disulfide bond between Cys99 and Cys256, the only two cysteines in each monomer (Figure 4 3). The disulfide bond was formed spontaneously, most likely during the oxidative folding process in the periplasm of E. coli (83). There is little to no information available on the oxida tive folding of T. cru but it is expected that the gammaproteobacterium have the machinery and conditions necessary to form disulfide bonds as part of protein folding. This disulfide bond connects a helix at the N terminal segment with a loop containing T hr252, which is part of the hydroge n bond network essential for CO 2 hydration (84). Superimposition of the active site of T. cru CA with hCA II (Figure 3 4) reveals that
66 Thr252, corresponding to Thr199 in hCA II, along with the other residues in the loop are aligned almost perfectly. Protein folding studies of hCA II have demonstrated that the conformation of the 198 206 loop is strained at Pro202, due to the adoption of a cis conformation that could potentially be stabilized by interactions with the resi dues at the 123 139 helix (80, 85). However, in T. cru CA, the segment corresponding to the 123 139 helix in hCA II is replaced with a short loop. Therefore, the presence of the disulfide bond could potentially stabilize the loop w ith residues Thr252 and Pro254. The same intramolecular disulfide bond corresponding to the linkage by Cys99 and Cys256 in T. cru CA monomers, such as hCA IV, hCA VI, hCA IX, hCA XII, NGCA, and Ao CA (12, 86 89, 100). The presence of the disulfid e bond in hCA IV was proved to confer the decreased susceptibility of the protein to denaturation by 5% SDS (90, 91). Guanidine HCl (GdnHCl) induced denaturation experiments performed with NGCA also demonstrated that the inactivation of NGCA occurred at lo wer concentrations of GdnHCl upon addition of the reducing agent tris(2 carboxyethyl)phosphine (TCEP), decreasing from 2.1 M GdnHCl to 1.2 M GdnHCl, which is comparable to the inactivation of hCA II by GdnHCl (at 0.9 M GdnHCl) (92, 93). The reduction of th e disulfide bond in NGCA led to decreased conformational stability, making it more sensitive to denaturation/inactivation by GdnHCl. It was inferred in CAs that the presence of the disulfide bonds accounted for increased conformational stability. Disulfide bonds in other globular proteins, such as Ribonuclease I, have also been linked to increased stability when compared to the reduced proteins (94 96). Furthermore, a study using an hCA II variant with an engineered disul fide bond at residues Cys23 and Cys203, equivalent to the
67 CA monomers and in T. cru CA, dramatically increased the conformational stability of hCA II by 3.7 kcal/mol, as calculated from an increase in the u nfolding midpoint from 0.9 to 1.7 M GdnHCl (97). DSC studies were performed as part of this study to determine the thermal stability of T. cru CA. From the scans we collected at pH 5 9, we observed that the melting temperature (T M CA went from 68.1 to 71.9C as the pH was increased, establishing a linear relationship between pH and temperature. The melting temperature of hCA II, also equal to its thermal inactivation temperature, is 59.5 0.5 C at pH 7.8 (78). However, we decided to set up a control of hCA II in the same buffer as T. cru CA, in order to allow direct comparison at pH 8.0. Our results show that at pH 8.0, T. cru CA has a T M of 71.9 0.4C, whereas hCA II has a T M of 55.96 0.05C. The 15 .94C difference between both T M demonstrates that T. cru CA has greater thermal stability than hCA II. Furthermore, the linear relationship between pH and temperature is not observed for hCA II (data not shown). Thermostability has long been associated with the structural and conformat ional stability of proteins, along with amino acid composition, hydrogen bonding and solvent accessibility (98, 99). By obtaining a larger T M T. cru CA is more thermodynamically stable, and therefore has greater confor mational stability than hCA II. Com parison of hCA II and T. cru CA structures revealed that there are minor structural differences, particularly shorter/missing surface loops and small changes in secondary structure. Having shorter surface loops provides T. cru CA with a slightly more c ompact structure, which could certainly enhance the folding stability. There could be other factors influencing the enhanced stability observed in T. cru CA, such as bulk
68 solvent interactions and hydrogen bonding networks, as well. However, we believe th at the disulfide bond present between Cys99 and Cys256 is the major contributor of the increased conformational stability of T. cru CA. The Dimeric Interface The crystal structure for T. cru CA contained 4 molecules in the asymmetric unit, which appeared to be interacting in pairs as a dimer or possibly a dimer of dimers. In order to determine the multimeric nature of the protein, we proceeded to use size exclusion chromatography to estimat e the molecular weight of T. cru CA. The protein eluted with an approximate molecular weight of 70 kDa, two times the molecular weight of the monomer, suggesting that the protein is a dimer in solution. Further evidence pointing to a dimeric interface wa s obtained from the DSC studies, which revealed two transition events, at 58C and 71C, representing the dissociation of the dimer and the unfolding of the monomers, respectively. T. cru CA forms a homodimer in solution, (Figure 4 8), deviating from mos class CAs, which are mainly monomeric. Crystal structures have been reported for CA isoforms that are also homodimeric in nature, which are the human CA in the unicellular green alga Chlamydomonas reinhar dtii (Cr CA expressed in Aspergillus oryzae (AoCA) (87 89, 100, 101). For the purposes of this discussion, we will compare the dimer interface of T. cru CAs. We used the Protein Interfaces, Surfaces and Assemblies (PISA) server to analyze and compare all possible interfaces of T. cru CA, in order to discriminate between real interfacing structures and interfaces resulting from crystal packing (102). We also used the PIS A
69 server to identify the interacting residues at the dimer interfaces of T. cru CA and all CAs presented in this discussion. Table 4 1 presents all possible interfaces present in the crystal structure of T. cru CA (Figure 4 9). The PISA server provides a list of parameters that ascertain the likelihood of a real interface interaction. For example, PISA provides the symmetry operators of each interface, which describe the symmetry operation applied to one of th e monomers in order to form the designated interface. In other words, the symmetry operators state how the monomer is moved in order to form the interface. From the results stated in the table regarding the symmetry operators, it is clear that the interfac es between chains B D, A D, and B C are not real interacting interfaces, as the interfaces created by these monomers are formed after applying a symmetry operator different to x, y, z. Other parameters used to discriminate between real and artificial interfaces are solv ) upon formation of the interface, the P value solv and the complexation significance score (CSS). The P value solv can basically be regarded as a measurement of inte rface specificity; the lower the P value is, the greater the probability that the interface is interaction specific. The CSS value, on the other hand, indicates how significant the interface interactions are for the association of the dimer on a scale of 0 1, where 0 implies that the interface is a result of crystal packing and 1 implies that the interface plays an essential role in complex formation. The results obtained from PISA demonstrate that the largest positive protein affinity is at the interface s formed by chains A B and C D, solv when the interface is formed. Also, the lowest P
70 values are observed for interfaces A B and C D. Furthermore, the CSS values for the interfaces A B and C D are near 1, indi cating that the interactions at these interface are significant for the dimer association. On the other hand, the interface between A solv and the P score, has a CSS value of 0.000, indicating that the interface is only a result of crystal packing, which is supported by the data obtained by gel filtration and DSC that there is only a dimer present in solut ion, and not a dimer of dimers. We also wished to analyze the residue specific interactions at the dimer interfac e of T. cru CA, and compare these to the residue specific interactions observed at the CAs in order to determine if the same interface is observed in any other isoform. Table 4 2 lists the hydrogen bond interactions observe d at the T. cru CA dimer interface. Note that the table only refers to chains A and B in the crystal structure, but the same residue specific interactions are also observed at the interface of dimer C D. Out of the 18 hydrogen bond interactions observe d at the dimer interface, 11 of them involve ionizable residues, which are Asp304, Lys246, and Glu258. Increasing the pH of the protein sample could ionize all three amino acids and strengthen the interaction formed by these residues. Although the pK a of l ysine is around 10 when exposed to free solvent, there is reason to believe that the actual pK a of Lys246 can be lower, due to the microenvironment formed by surrounding residues (Figure 4 10). Microenvironments formed in active sites, protein pockets, or at interfaces a (103). In T. cru CA, the ionizable residues are found buried at the dimer interface, where Lys246 is almost completely buried (90%), and Asp 304 and Glu258 are 50% and 70% buried, respectively. Intera ctions involving
71 surrounding residues, some of which are hydrophobic, could decrease the pK a of Lys148, effectively deprotonating the Lys148 upon increase of the overall pH. Therefore, involvement of ionizable groups at the dimeric interface can explain th e increase in the T M of the dissociation of the dimer, as the hydrogen bond interactions at the interface are strengthened when pH is increased from 5 to 9. Hydrophobic residues were also found to interact at the dimer interface. The major hydrophobic resi dues involved in residue specific interactions at the interface are Val116, Leu121, Gly259, Val260, and Leu299. Interestingly, Val260 is also part of the hydrophobic patch at the active site of T. cru CA, corresponding to Val(207) in hCAII, and is involv ed in CO 2 binding (2, 104). The side chain of Val260 is displaced 1.2 away from the active site toward the CO 2 binding site, possibly due to its involvement at the dimer interface (Figure 3 4). The displacement of Val260 could potentially affect the CO 2 binding orientation, which could decrease the effectiveness of the nucleophilic attack by the zinc bound hydroxide. Therefore, the interaction of Val260 at the dimer interface could explain the reduced catalytic efficiency of T. cru CA. We compared the d imer interface of T. cru CA to the interface of the other CAs by superimposition of the dimeric structures and PISA analysis (Table 4 CAs, the interactions at the dimer interface o f T. cru CA dictate the formation of the dimer. However, the lower CAs do not imply that the association of the dimers is a result of crystal packing, as they have been proved to be dimeric in nature, but that the inte rface of the dimers is of a more hydrophilic nature (102). Except for hCA CAs compared in this study exceed the 860 2 cutoff value of surface
72 area buried per monomer used to discriminate between monomers and homodimers (105). However, hC A IX, along with Cr Cys41 bridging the two monomers together. Therefore, a reason why these have CSS values closer to 0, while still being classified as dimers, is because the assembly of the complex depends gr eatly on the formation of the intermolecular disulfide bond. CAs by superimposition of the structures on T. cru CA using Coot (Table 4 4). Coot was not able to superimpose CAs onto T. cru CA, superimposing instead only one of the monomeric units for each structure. Interestingly, we found that T. cru CA forms a unique dimer interface, which is why Coot wa s not able to accurately superpose the dimeric unit without distorting the secondary structures. The T. cru CA dimer interface is formed mostly by interactions of residues at the N and C terminus, with each monomer related by a 2 fold symmetry axis at t he interface. Figures 4 11 to 4 15 illustrate the dimer interface of hCA VI, hCA IX, hCA XII, Cr Ao CA in relation to the interface of T. cru CA. Additionally, all residues forming part of the interface of the dimeric CAs are listed in Table 4 5. CAs demonstrate different residue specific interactions involved in the association of the dimer. Furthermore, superimposition of all dimers onto T. cru CAs share the same interface (Figure 4 CA interface that most closely resembles the orientation of the dimer association of T. cru CA is hCA IX, where even some residues appear to overlap in the interface. However, most of the residues th at
73 are involved in the dimer association of hCA IX do not form part of the interface of T. cru CA.
74 Table 4 1. Possible interfaces observed in the crystal structure of T. cru CA as determined by PISA (102) Interfaces Symmetry operator a Interface Area b 2 No. Interfacing Residues c solv. d kcal/mol solv. P Value e CSS f A B x, y, z 897.9 63 (31 32) 9.7 0.123 0.963 C D x, y, z 882.6 66 (34 32) 8.6 0.176 0.963 A C x, y, z 529.5 33 (17 16) 5.7 0.197 0.000 B D x y + z 506.8 35 (18 17) 5.6 0.197 0.000 A D x + 3/2, y z +2 155.3 14 (6 8) 0.9 0.480 0.000 B C x + 2, y, z + 3 261.5 20 (5 15) +1.1 0.684 0.000 a Symmetry operation applied to the 2 nd interfacing structure to obtain the interface, specified in fractional space relative to the initial structure position. b Calculated as difference in total accessible surface areas of isolated and interfacing structures divided by two. c Values in parenthesis correspond to number of residues from each monomer. d Solvation free energy gain upon formation of the interface. solv < 0 corresponds to or H bon ds across the interface). e Measure of interface specificity. f Indicator of interface significance for assembly formation.
75 Table 4 2. Hydrogen bond interactions at the T. cru CA dimer interface. Monomer A Monomer B Distance () Asn104 O Ser248 O 3.6 Ala115 O Ser120 O 3.9 Gly117 O Thr119 N 3.1 Thr119 O Asp304 N 3.0 Ser120 O Gly117 N 3.3 Glu258 O Asn104 N 3.6 Ala295 O Lys246 N 2.5 Asp304 O Ser120 O 2.7 Asp304 O Ser120 N 3.8 Asp304 O Thr119 O 2.3 Ser248 O Asn104 O 3.2 Thr119 N Gly117 O 3.3 Asp304 N Thr119 O 3.0 Gly117 N Ser120 O 3.4 Asn104 N Glu258 O 3.4 Lys246 N Ala295 O 2.4 Ser120 O Asp304 O 3.2 Thr119 O Asp304 O 3.1
76 Table 4 3. Comparison of the interfaces of T. cru CAs Dimeric CA Interface Area a 2 No. Interfacing Residues b solv. c kcal/mol solv. P value d CSS e No. Hydrogen Bonds T. cru CA 897.9 63 (31 32) 9.7 0.123 0.963 18 hCA VI 869.3 54 (28 26) 9.8 0.552 0.000 11 hCA IX 801.6 54 (27 27) 9.6 0.170 0.000 2 hCA XII 1130.2 72 (35 37) + 1.1 0.783 0.000 17 Cr 1493.3 84 (42 42) 17.7 0.068 0.221 21 Ao CA 1812.2 93 (46 47) 18.4 0.189 0.265 23 a Calculated as difference in total accessible surface areas of isolated and interfacing structures divided by two. b Values in parenthesis correspond number of residues from each monomer. c Solvation free energy gain upon formation of the interface. solv < 0 corresponds to or H bonds across the interface). d Measure of interface specificity. e Indicator of i nterface significance for assembly formation.
77 Table 4 4. Comparison of dimer interfaces by secondary structure matching (SSM) superposition of T. cru CA using Coot (64) Sequence identity (%) No. aligned residues Bond length RMSD () hCA VI (3FE 4) 32.52 206 1.740 hCA IX (3IAI ) 35.50 214 1.374 hCA XII (1JCZ ) 33.18 217 1.426 Cr ) 33.48 221 1.611 Ao CA (3Q 31) 29.47 207 1.606
78 Table 4 5. List of interfacing residues of dimeric CAs Residue type T. cru CA hCA VI hCA IX hCA XII Cr Ao CA Hydrophilic Asn104 Ala115 Gly117 Thr119 Ser120 Ser248 Lys246 Glu258 Ala295 Asp304 His38 Tyr39 Glu77 Pro79 Ser90 Asp150 Gln153 Asp154 Tyr189 Pro190 Gln192 Arg193 Thr194 Thr195 Pro223 Ser41 Pro42 Pro84 Arg86 Tyr88 Ser124 Glu133 Arg137 Pro138 Glu195 Arg254 Glu257 Tyr7 Glu13 Asn14 Ser15 Ser17 Lys18 Cys23 His34 Asp36 Asn99 Asp102 His103 Ser110 Gln112 Asn243 Arg246 Gln274 Gln249 Lys150 Asp252 Cys21 Lys24 Asn45 Asn71 Thr167 Lys175 Tyr177 Pro178 Gln207 Asn210 Asn285 Arg288 Gln358 Asn362 Pro363 Tyr365 Thr40 Asn47 Tyr49 Glu53 Thr126 Pro127 His131 Glu134 Glu135 His136 Pro138 Gln160 Glu163 Tyr239 Asn240 Lys243 Lys247 Tyr248 Tyr252 Thr253 Gln254 Asn255 Glu264 Lys269 Hydrophobic Val116 Leu121 Gly259 Val260 Leu299 Ile151 Gly191 Phe27 Ala39 Ala43 Gly85 Ala127 Phe128 Gly136 Gly139 Gly253 Val255 Phe8 Gly9 Gly24 Ile37 Gly111 Ile242 Phe245 Lys248 Ile22 Gly43 Leu44 Leu171 Ala173 Gly174 Ile176 Leu208 Phe281 Trp284 Leu293 Val354 Ala355 Phe356 Phe360 Gly41 Leu42 Leu46 Val68 Phe99 Leu161 Val236 Leu262 Leu263 Val265 Ala266 Ala267 Leu270 Cystines Cys41 Cys21
79 Figure 4 1. Representation of the crystal packing of T. cru CA. A unit cell (denoted by the blue box) for T. cru CA contains 16 chains, 4 molecules per asymmetric unit (au). Each molecule in the au is depicted in a different color.
8 0 Figure 4 2. Ramachandran plot of T. cru CA residues obtained from PROCHECK (66). The Ramanchandran plot statistics show that over 90% of the dihedral angles of T. cru CA are within the most favored regions, while the rest are in the additionally allowed regions, except for 1.0% of the angles that fall within the generously allowed regions.
81 Figure 4 3. Cartoon representation of T. cru CA. The structure is oriented in such a sheet fold CAs can be seen better. Zinc coordinating histidines and the disulfide bond represented by sticks. Nitrogen atoms are shown in navy blue, and sulfur atoms in yellow. Zinc ion is shown as a gray sphere.
82 Figure 4 4. Close up stick representation of the T. cru CA active site. Residues Tyr80, Asn138, His140, Thr141, Gl n143, Glu171, and Thr252 form the hydrophilic side of the active site (yellow) which aids in proton transfer and bicarbonate removal, while residues Val186, Val196, Leu251, Val260, and Trp262 line the hydrophobic side (blue), also the CO 2 binding site. Nit rogen atoms are shown in navy blue; oxygen in red, and the zinc ion as a gray sphere.
83 Figure 4 5. Comparison of T. cru CA (green) and hCA II (gray) (PDB ID: 3KS3; 54). The deletion of surface loops in T. cru CA lead to a structure with a wider gap at the active site cavity, increasing solvent accessibility. Zinc ion is shown as a gray sphere.
84 Figure 4 6. Sequence alignment of hCA II and T. cru CA determined by ClustalW (106, 107). The sequence alignment demonstrates the additional N terminal region formed by residues 23 74 in T. cru CA, as well as the predicted gaps in the T. cru CA sequence compared to hCA II.
85 Figure 4 7. Disorder probability plot obtained from PONDR FIT (81). The plot shows that some regions, particularly in the N terminus and at the C terminus, are probably disordered. Disorder in some regions could cause the absence of these and adjacent regions in crystal structures.
86 Figure 4 8. Cartoo n representation of the dimer formed by chains A (left) and B (right) of T. cru CA. The monomers in the homodimer are related by a 2 fold axis along the N and C terminus. The zinc coordinating histidines and the disulfide bond are shown as sticks. Nitro gen atoms are shown in navy blue, sulfur atoms in yellow, and the zinc ion as a gray sphere.
87 Figure 4 9. Possible interfaces present at the crystal structure of T. cru CA. The PISA server (102) analyzed the interfaces that could potentially form between the chains within the au of T. cru CA. The black circles drawn above represent some of the possible interfaces. AB, AC, CD, and BD represent the interface formed bet ween chains A and B, chains A and C, chains C and D, and chains B and D, respectively.
88 Figure 4 10. Buried lysines at the T. cru CA dimer interface. The microenvironment formed by the surrounding hydrophobic residues Val 116, Leu121, and Leu299 can affect the pK a of the buried lysines. Nitrogen is shown in blue, and oxygen in red.
89 Figure 4 CAs T. cru CA (green) and hCA VI (teal) (PDB ID: 3FE4; 100). The dimer interface of hCA VI is very different from that of T. cru CA, as it appears to be right on top of the active site cavity. Zinc ion is shown as gray sphere. Interface residues are shown as sticks. Nitrogen atoms are shown in blue, sulfur in yellow, and oxygen in red.
90 Figure 4 12. Superimposition of T. c ru CA (green) and hCA IX (orange) (PDB ID: 3IAI; 87) dimeric structures. Some residues involved in the dimer association of hCA IX appear in the interface of T. cru CA as well. Nitrogen atoms are shown in navy blue, sulfur atoms in yellow, oxygen atoms in red, and the zinc ions are represented by gray spheres.
91 Figure 4 13. Superimposition of T. cru CA (green) and hCA XII (blue) (PDB ID: 1JCZ; 88). The dimer interface of T. cru CA and hCA XII are also very different. Nitrogen atoms are s hown in navy blue, sulfur atoms in yellow, oxygen atoms in red, and the zinc ions are represented by gray spheres.
92 Figure 4 14. Superposition of T. cru CA (green) and Cr interfaces of T. cru CA and Cr atoms in yellow, oxygen atoms in red, and the zinc ions are represented by gray spheres.
93 Figure 4 15. Superimposition of T. cru CA (green) and Ao CA (c yan) dimers (PDB ID: 3Q31; 89). The residues at the dimer interface of T. cru CA and Ao CA differ, leading to a different orientation of dimer association. Nitrogen atoms are shown in navy blue, sulfur atoms in yellow, oxygen atoms in red, and the zinc io ns are represented by gray spheres.
94 Figure 4 CAs on T. cru CA (green). On the figure to the right, it can be observed CAs superimposed well on one of the monomers. However, none of the dimers superimpose on each other, so all of the dimers form a unique interface. The view has been altered for clarity. Zinc ions are shown as gray spheres.
95 CHAPTER 5 CONCLUSIONS CA expressed in the T. cru gammaproteobacterium provided an insight to increase thermal CAs that could be used for industrial applications, such as CO 2 scrubbers or a rtificial lungs. T. cru class CAs that shows increased thermal and pH CAs. Results from this study, along with studies performed with thermostable CAs NGCA and hCA IV (90 93), suggest that the disulfide bond present between residues Cys99 and Cys256 is the major contributor of the increased thermal stability of T. cru CA. The disulfide bond, along with the pH independent stability observed between pH values 5 9, are adaptati ons that allow the T. cru CA to remain fully functional in the erratic environment that dominates the hydrothermal vents, and could potentially be used to engineer CAs with increased thermal and pH stability. Further studies need to be completed to deter mine the structural or physical features that stabilize the protein at different pH values. class CAs, T. cru CA forms a homodimer in solution. Comparable to the melting temperature of the unfolding of T. cru CA, the temperature corresp onding to the dissociation of the dimer was directly proportional to the pH. An increased dimer association, which would result in a greater T M suggested the presence of ionizable groups at the interface. A closer look at the interfacing residues reveale d a buried lysine (Lys246) could have a decreased pK a that would result in amine group at lower pH, enhancing the hydrogen bonds present at the interface. Comparison of the dimeric interface of T. cru
96 CAs de monstrated that T. cru CA forms a unique interface. Furthermore, we observed CAs used for comparison in this study share the same interface. The crystal structure of T. cru CA lacks 52 amino acid residues at the N terminus, a region that appears to be unique among known proteins to date. The region is of highly hydrophilic content with a basic nature that could potentially affect the nucleophilicity of the zinc bound water by decreasing its pK a resulting in a decreased catal ytic efficiency as compared to hCA II. Since the 52 N terminus residues were missing in both the crystal structure and previous homology models, we assessed the possibility of an intrinsically disordered region within T. cru CA and observed that the N terminus was predicted to be partially disordered. Additional studies could be performed in order to further understand the disordered nature of the missing N terminal region and determine if this region might be involved in o ther biological functions. Overall, the T. cru CA is comparable to hCA II in structure and catalytic activity, with major differences in the thermal and pH stability. The similarities in structure and activity to hCA II suggest that the biological roles of T. cru CA are fairly similar to those of hCA II. Therefore, T. cru CA could very well be involved in CO 2 transport within the periplasm to the cytoplasm and pH homeostasis. T. cru CA could also be involved in sequestering the CO 2 and bicarbonate i n the periplasm to prevent CO 2 from diffusing out of the cell. The differences in thermal stability between T. cru CA and hCA II suggest that the enhanced thermal and pH stability of the T. cru CA is vital for the growth and survival of T. cru
97 In con clusion, this project describes the structure, catalysis, and biophysical nature of the T. cru CA within the T. cru gammaproteobacterium. Several roles have been suggested, and will require whole cell studies to confirm the physiological relevance of these roles. Additional studies are also required to structurally, kinetically, and biophysically characterize the other CAs expressed in T. cru Characterization of the other CAs, along with who le cell studies, can provide the information needed to assess the biological interactions of these CAs and determine their physiological functions, as well as the relevance of the biological interactions to the growth and survival of T. cru
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108 BIOGRAPHICAL SKETCH Natalia has been involved in research since she was a sophomore at the to Research Careers Undergraduate Student Training Award. She used to work with snake venom phospholipases A 2 under the guidance of Dr. Reginald Morales. During her junior year, she completed a summer internship at Brown University as part of the Leadership Alliance Summer Research Early Identification Program, and worked with Dr. Kimberly Mowry, studying Vg1 mRNA localization in Xenopus laevis t the conclusion of her internship, she presented her projects at two national meetings. Natalia graduated from the University of Puerto Rico with a Bachelor of Science in Chemistry, and was awarded Magna c um Laude honors. Natalia joined the McKenna labora tory in the spring of 2011. Her thesis project was based on the characterization of an carbonic anhydrase expressed in the deep sea vent Thiomicrospira crunogena gammaproteobacterium. During her leisure time, Natalia volunteered at a local hospital a few hours a week, and played catcher for the aspires to venture into a career in medicine.