Structural and Kinetic Analysis of Specific PI-3 Mutants of the C-terminal Polyproline Helix

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Structural and Kinetic Analysis of Specific PI-3 Mutants of the C-terminal Polyproline Helix
Jackson, Larry
Dunn, Ben ( Mentor )
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Gainesville, Fla.
University of Florida
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Structural and Kinetic analysis of specific PI 3 mutants of the C
terminal Polyproline Helix

Larry Jackson


Ascaris suum is a nematode that primarily infects pigs but also causes disease in humans. The parasite carries

out part of its life-cycle in the intestinal tract of the host where it is subject to the harsh environment of the

stomach, including exposure to the digestive enzymes pepsin and gastricsin. As a part of its survival mechanism,

the worm produces a 17KDa protein, pepsin inhibitor-3 (PI-3) (Abu-Erreish and Peanasky, 1974). Recombinant PI-

3 expressed in E. Co// has been previously shown to be a competitive inhibitor of a sub-group of aspartic

proteinases: pepsin, cathepsin E, and gastricsin. From the crystal structure of the complex of PI-3 with porcine

pepsin (p. pepsin), we know that the first three N-terminal residues and a polyproline (139-142) helix in the

C-terminal domain of PI-3 are points of contact between the inhibitor and p.pepsin (Ng et al, 2000). We have

made proline to alanine mutations in the polyproline helix and have performed kinetic studies to evaluate

the significance of this region for inhibition of porcine pepsin and the malarial enzyme plasmepsin 2. We

monitored these changes in inhibition for the mutants as measured K, values. When comparing the K, values for

the PI-3 mutants to that of the wild type inhibitor using porcine pepsin and a chromogenic substrate

(KAIEF*NphRL), there is no significant change for the single mutant P141A. However, for the mutants P140A/

P141A and P139A/P140A/P141A there is significant increase in the K,, although they are still tight-binding

inhibitors with K, values 2.8 nM and 3.OnM, respectively. However for plasmepsin 2, all PI-3 mutants have a

K, significantly larger than wild-type except for the P140A/P141A mutant.


Aspartic proteinases encompass a variety of proteolytic enzymes that are characterized by having acidic

isoelectric points and maximal activity in acidic environments. Aspartic proteinases predominantly target

substrates containing hydrophobic residues in the P1 and Pl' positions. Aspartic proteinases have two conserved

Asp-Thr/Ser-Gly sequences, one in each domain. Peptide bond hydrolysis is catalyzed by two aspartate

residues juxtaposed to one another. One of the residue side chain carboxylate groups is depronated at the

favored acidic conditions of these enzymes and acts as a proton acceptor from a water molecule in the active

site. Simultaneously, the water performs a necleophilic attack on the carbonyl carbon of the P1 position,

disrupting the scissile bond to the Pl' residue.

The aspartic proteinases are involved in a number of important biological and physiological processes (Fowler et

al., 1995). In animals, the enzyme renin has a hypertensive action through its role in the renin-angiotensin

system (Davies, 1990). The retroviral aspartic proteinases, such as HIV proteinase, are essential for the

maturation of the virus particle (Vogt, 1996). The lysosomal aspartic proteinase cathepsin D has been implicated

in tumorigenesis. The stomach enzyme pepsin, which plays a major physiological role in hydrolysis of acid-

denatured proteins, is responsible for much of the tissue damage associated with peptic ulcer disease (Cooper, 2002).

Ascaris lumbricoides, a parasitic nematode, is one of the largest and most common parasites found in humans.

This particular species of worm, estimated at infecting 25% of the world's population, completes its life cycle

within the small intestines and is the cause of significant pathology to the lungs and gastrointestinal systems of

its host ( A closely related counterpart Ascarls suum, found in

pigs, can cause similar pathology within a human host. The majority of these infections occur in tropical regions

of the world where high population density and poor sanitation are prevalent. Ascaris infections typically

affect children, and the presence of the worms can contribute to retardation of physical development and malnutrition.

Both Ascaris worms are exposed to digestive enzymes of their host and in response to their colonization within

the harsh environment of the intestinal tract, the worms produce several proteinase inhibitors that the assist

the organisms' survival in the digestive tract (Abu-Erreish and Peanasky, 1974). These proteinaceous inhibitors

show specific affinity toward carboxypeptidase's A and B, trypsin, chymotrypsin, and pepsin. Many of these

proteins are produced in large numbers in the digestive tract of the worms to protect the worms from the digestion

of cell surface proteins and regulation of nutritional requirements. Several proteinase inhibitors from the

Ascaris suum species, most notably pepsin inhibitor-3, have been characterized and shown to inhibit the

Journal of Vndergradllatc Researcl: University of Florida

enzymes pepsin, gasticsin, and cathepsin E. PI-3 had weak affinities and no detectable inhibition for fungal

enzymes and cathepsin D.

The structure of the Ascaris suum pepsin inhibitor 3 and its subsequent complex with porcine pepsin (p. pepsin)

has, revealed the workings of this novel mode of competitive inhibition on proteins within the aspartic

proteinase family (Ng et al., 2000). PI-3 adopts a novel fold consisting of two ant parallel 3-sheets, each flanked by

a single helix. The molecule has the shape of a flat, rectangular box and can be divided into two domains. The

C-terminal domain contains the polyproline II helix, residues (139-142) that packs against a helix II (Ng. et

al., 2000). Currently, studies are being performed to test the characteristics of recombinant PI-3 mutants

with porcine pepsin in order to ascertain structural and kinetic data that will one day lead to new techniques

in designing protein inhibitors for the specific family of proteins.

The focus of my research project will involve structural and kinetic assays towards the C-terminal domain of PI-

3, which contains the five proline residues that form a polyproline helix that makes van der Waals interactions with

a specific C-terminal loop of porcine pepsin. In an attempt to alter specificity, the five proline residues will

be mutated to alanine residues to somewhat relax the specificity. Mutations, through QuikChange mutagenesis,

will begin with the central alanine residue (~PPAPP~), and then two double mutants will be made with the

central alanine and one other on either side (~PAAPP~, ~PPAAP~). Finally, mutants will be made where

three residues on either half are altered (~AAAPP~, ~PPAAA~). Kinetic analysis with a chromogenic substrate will

be made of the single, double, and triple mutant in order to determine the importance of the five positions in

binding to porcine pepsin. With these specific PI-3 mutants, studies will be conducted with related enzymes within

the aspartic proteinase family to test inhibition efficiency.

The data obtained from the kinetic analysis of mutant PI-3 will not only provide valuable insight into the

optimal inhibition with PI-3, but this information will be applied to another class of enzymes within the

aspartic family, the aspartic endopeptidases (plasmepsins). Malaria, caused by several protozoan species of

the genus Plasmodium, is responsible for the deaths of millions of people each year. Two aspartic

proteinases (plasmepsin I and plasmepsin II) of the human malarial parasite Plasmodium falciparum play key roles

in the essential pathway by which the parasites catabolise up to 80% of hest cell hemoglobin in order to

obtain critical amino acids for protein synthesis. Hopefully, data obtained from the experiments with PI-3 will help

to improve the inhibition of plasmepsin 2.



Mutations will begin with the central alanine residue (~PPAPP~), and then two double mutants will be made with

the central alanine and one on either side (~PAAPP~, ~PPAAP~). Finally, mutants will be made where three

residues on either half are altered (~AAAPP~, ~PPAAA~). Mutations were added using the QuikChange Site

Directed Mutagenesis Kit (Stratagene).

Protein expression and Inclusion Body Isolation.

The recombinant PI-3 mutants were purified from the over-expression of the inserted gene in BL21-

DE3 pLysS cells that have been transformed with pET-3d vector.

Protein Refolding.

Cells were lysed using a SLM-Aminco French Pressure cell at a 1000 psi. Inclusion body pellet

was retrieved by centrifuging the cell lysate over 10 mLs of 27% sucrose in 30ml Corex tubes in a

JS-14.1 swinging bucket rotor at 8,000 x g for 45 minutes. The supernatant was immediately

decanted and a sample taken to run on an SDS-PAGE gel. The inclusion body pellet was resuspended in

a total of 20mis of TN-Triton buffer. The suspension was layered over 10mIs of 27% sucrose and

spun down as previously described to pellet the inclusion bodies. The resulting inclusion bodies

will resolubilized using an SM-urea solution mixed with Amberlite ion exchange resin,

1S-mercaptoethanol, and CAPS buffer to denature all insoluble proteins. The dissolved protein

will dialyzed with a 6-8 kDa molecular weight cut off tubing submerged in a variety of buffers

including MOPS and Tris buffers at varying pH's.

Ammonium Sulfate Precipitation.

To concentrate the recombinant PI-3 mutants, the post-dialysate was subjected to ammonium

sulfate ((NH4)2S04) precipitation. The post-dialysate was stirred on ice and (NH4)2SO4 was slowly

added to 40% saturation and allowed to stir for one hour. The solution was centrifuged at 12,500xg

for 45 minutes. The precipitate was dissolved in sodium phosphate buffer and transferred to a

15mL conical tube. The above procedure was repeated for 70% ammonium sulfate saturation.

The resuspended precipitates from the 40% and 70% ammonium sulfate fractions in the 15 ml

conical tubes were spun down in a Beckman GS-15R centrifuge with Beckman rotor S4180 at 4800 rpm

to remove any precipitate that does not re-dissolve. The supernatant was transferred to a 15 mL

conical tube. The precipitate was resuspended in sodium phosphate buffer and a sample taken to run

on an SDS-PAGE.

Spectrophotometric Analysis of Enzyme activity.

Assays will be done on a Hewlett Packard 8452 A diode array spectrophotometer. Initially, reactions

will be carried out using porcine pepsin with a chromogenic substrate to obtain Ki values on the

different mutants to determine the importance of the five positions in binding to either human or

porcine pepsin.


Table 1
Proline to alanine mutations of the polyproline helix (residues 139-142) of PI-3. Mutations were made with the
QuikChange Mutagenesis Kit from Stratagene

Mutations to Polyproline Helix
wt 139 140 141 142 143

P141A P P P P P

P140A/ A A P P


P14OA/ A A A P P

Table 2
K1 values in nM for wild type P13 and polyproline helix mutants with p.pepsin and plasmepsin 2

Kinetic Analysis of P13

Type Porcine Pepsin Plasmepsin 2
Wild Type 019 +004 85 + 9

P141A 031 +008 140+20

P140A/P141A 28+05 70+ 8

P141A/P142A 053 +006 106+ 10

P139A/P140A/P141A 30+07 120+ 13



E quations:

Tight binding inhibiton. vo = V_ /(K(/S+l)K,~=Ki*((S/KIR+I)


Classic inhibition v=V-,1(1+(Km/S)*(l+I/f,))


For porcine pepsin, kinetic analysis shows that while mutations to the polyproline helix increase the K1, the

P13 mutants are still considered tight binding inhibitors. This study also indicated that mutations to the N-

terminal side of the central proline (P141) are more detrimental to the potency of the inhibitor than mutations on

the C-terminal end of the polyproline helix. For plasmepsin 2, the mutations were not able to significantly lower

the K1. I have also shown that the mutation to P140 together with P141A seems to compensate for the

negative effects of the single P141A mutation. This is a different pattern from p.pepsin and may indicate

different points of contact between enzyme and inhibitor. In addition, inhibitor constant determinations

were performed with mutant P143 and porcine pepsin. The K1 value marginally increased with the P143A mutant

as compared to the P139A/P140A/P141A triple mutant.

Figure 1. Ribbon diagram of crystal structure of p.pepsin (blue/green) completed with PI-3

(yellow). Arrow points to area where the polyproline helix (139-143) interacts with p.pepsin

"290's loop" (290-294).



D290 81381

Figure 2. Polyyproline helix of PI-3 completed with porcine pepsin. James, MNG., et al Nature,


Alignment of P. pepsin with Plasmepsin 2

288 292 298
P. pepsin G M D V P T S S G E L

lasmepsin 2 G L D F P - - - V P T

P143A P P P P

Figure 3. Connolly surface showing polyproline helix of PI3 (yellow), residues 288-298 of p.

pepsin (blue) and plasmepsin 2 (red). The plasmepsin 2 has been superimposed onto the

structure of p. pepsin completed with PI3, determined from x-ray crystallography. The conserved

residue P292 for each enzyme is indicated by the arrows.


3 97 + 0 6

.* .*
* t

j so

* D * *

3D 31 V 33 3$ 36 37! Ms 3 0 41 42 0& - 45 0 6 0 7 .0 49 5R 61 52

Figure 4. Graph plotting the fraction numbers as designated from fraction collection tubes versus

the % inhibition of porcine pepsin with PI3 fraction. Fractions 38-43 contain the highest %


% itlhbmtin of P-ri.ii P� pm with P13 FraL-tio-




20 40 S 0 OM
[Substrate] (pM)

* 1-D
l I "10
Figure 5. Inhibitor dissociation constant (Ki) determination. Michaelis-Menten curve fit of rate

(pmol/min/mg) versus [substrate] with increasing inhibitor concentrations ([I]).

Figure 6. Line weaver-Burk linear regression of the Michaelis-Menton plot showing competitive



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Ng, K. K., Petersen, J. F., Cherney, M. M., Garen, C., Zalatoris, J. J., Rao-Nalk, C., Dunn, B. M., Martzen, M.

R., Peanasky, R. J., and James, M. N. (2000). Structural basis for the inhibition of porcine pepsin by Ascarins

pepsin inhibitor-3. Nat Struct Biol 7(8), 653-7.

Richter, C., Tanaka, T., and Yada, R. Y. (1998). Mechanism of activation of the gastric aspartic

protelnases: pepsinogen, progastricsin and prochymosin. Biochem J 335(Pt 3), 481-90.

Tang, J., James, M. N., Hsu, I. N., Jenkins, J. A., and Blundell, T. L. (1978). Structural evidence for gene

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