Structural and Kinetic analysis of specific PI 3 mutants of the C
terminal Polyproline Helix
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 (www.biosciohio-state.edu/~parasite/ascarls.html). 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.
MATERIALS AND METHODS
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.
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
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
P P A A P
P14OA/ A A A P P
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
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).
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
* 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 Pï¿½ pm with P13 FraL-tio-
20 40 S 0 OM
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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Douvres, F. W., Tromba, F. G., and Malakatis, G. M. (1969). Morphogenesis and migration of Ascaris suum
larvae developing to fourth stage in swine. J Parasitol 55(4), 689-712.
Fowler, S. D., Kay, J., Dunn, B. M., and Tatnell, P. J. (1995). Monomeric human cathepsin E. FEBS Lett 366(1), 72-4.
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
duplication in the evolution of the acid proteases. Nature 271(5646), 618-21.
Vogt, V. M. (1996). Proteolytic processing and particle maturation. Curr Top Microbiol Immunol 214, 95-131.
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