Structure-Based Optimization of Protease-Inhibitor Interactions to Enhance Specificity of Human Stefin-A against Falcipain-2 from the Plasmodium falciparum 3D7 Strain.

The emergence of resistance in Plasmodium falciparum to frontline artemisinin-based combination therapies has raised global concerns and emphasized the identification of new drug targets for malaria. Cysteine protease falcipain-2 (FP2), involved in host hemoglobin degradation and instrumental in parasite survival, has long been proposed as a promising malarial drug target. However, designing active-site-targeted small-molecule inhibitors of FP2 becomes challenging due to their off-target specificity toward highly homologous human cysteine cathepsins. The use of proteinaceous inhibitors, which have nonconserved exosite interactions and larger interface area, can effectively circumvent this problem. In this study, we report for the first time that human stefin-A (STFA) efficiently inhibits FP2 with Ki values in the nanomolar range. The FP2-STFA complex crystal structure, determined in this study, and sequence analyses identify a unique nonconserved exosite interaction, compared to human cathepsins. Designing a mutation Lys68 > Arg in STFA amplifies its selectivity garnering a 3.3-fold lower Ki value against FP2, and the crystal structure of the FP2-STFAK68R complex shows stronger electrostatic interaction between side-chains of Arg68 (STFAK68R) and Asp109 (FP2). Comparative structural analyses and molecular dynamics (MD) simulation studies of the complexes further confirm higher buried surface areas, better interaction energies for FP2-STFAK68R, and consistency of the newly developed electrostatic interaction (STFA-R68-FP2-D109) in the MD trajectory. The STFA-K68R mutant also shows higher Ki values against human cathepsin-L and stefin, a step toward eliminating off-target specificity. Hence, this work underlines the design of host-based proteinaceous inhibitors against FP2, with further optimization to render them more potent and selective.

New insights of falcipain 2 structure from Plasmodium falciparum 3D7 strain.

Malaria identifies as a tropical hallmark, conforming to the burgeoning notion of escalating drug resistance among virulent strains, with the burdensome Plasmodium falciparum under its wing. The cysteine protease Falcipain-2 (FP2) is released in the parasite's food vacuole in the trophozoite stage and contributes to disease progression through its hemoglobinase activity. In the present study, we have determined the crystal structure of FP2 from a drug resistant P. falciparum 3D7 strain. FP2-3D7 sequence has detected four amino acid variants, R12K, E14 N, P100T and G102D, in the mature domain of the protease, when compared with other reported structures. FP2-3D7 protease has been crystallized in the presence of two inhibitors E-64 and Iodoacetamide, which diffracted up to 3.5 Å and 3.4 Å respectively. Structural analyses of the mature domain helped unveil two solvent-exposed pockets with bound ligands where one is structurally homologous to the allosteric binding site of human Cathepsin-K and thus, could be exploited for designing allosteric modifiers of FP2. The structure has also revealed (poly)ethylene glycol molecules along the catalytic cleft, providing interesting insights for exploring FP2 as a chemotherapeutic target and for PEGylation in drug delivery. The side-chains of P2 and P3 residues of E-64 also adopt different rotamer conformations, compared with previously reported structure, emphasizing strain-specific multiple binding-modes of active-site targeted inhibitors. Docking studies, along with normal mode analyses, highlight the mode of hemoglobin binding and the active/inactive switch in hemoglobinase activity, conjecturing the formation of a stable dimeric state with a symmetry related copy in crystal packing.

Structure-guided protein engineering of human cathepsin L for efficient collagenolytic activity.

Engineering precise substrate specificity of proteases advances the potential to use them in biotechnological and therapeutic applications. Collagen degradation, a physiological process mediated by collagenases, is an integral part of extracellular matrix remodeling and when uncontrolled, implicated in different pathological conditions. Lysosomal cathepsin-K cleaves triple helical collagen fiber, whereas cathepsin-L cannot do so. In this study, we have imparted collagenolytic property to cathepsin-L, by systematically engineering proline-specificity and glycosaminoglycans (GAG)-binding surface in the protease. The proline-specific mutant shows high specificity for prolyl-peptidic substrate but is incapable of cleaving collagen. Engineering a GAG-binding surface on the proline-specific mutant enabled it to degrade type-I collagen in the presence of chondroitin-4-sulfate (C4-S). We also present the crystal structures of proline-specific (1.4 Å) and collagen-specific (1.8 Å) mutants. Finally docking studies with prolyl-peptidic substrate (Ala-Gly-Pro-Arg-Ala) at the active site and a C4-S molecule at the GAG-binding site enable us to identify key structural features responsible for collagenolytic activity of cysteine cathepsins.

Characterizations of SARS-CoV-2 mutational profile, spike protein stability and viral transmission.

The recent pandemic of SARS-CoV-2 infection has affected more than 3.0 million people worldwide with more than 200 thousand reported deaths. The SARS-CoV-2 genome has the capability of gaining rapid mutations as the virus spreads. Whole-genome sequencing data offers a wide range of opportunities to study mutation dynamics. The advantage of an increasing amount of whole-genome sequence data of SARS-CoV-2 intrigued us to explore the mutation profile across the genome, to check the genome diversity, and to investigate the implications of those mutations in protein stability and viral transmission. We have identified frequently mutated residues by aligning ~660 SARS-CoV-2 genomes and validated in 10,000 datasets available in GISAID Nextstrain. We further evaluated the potential of these frequently mutated residues in protein structure stability of spike glycoprotein and their possible functional consequences in other proteins. Among the 11 genes, surface glycoprotein, nucleocapsid, ORF1ab, and ORF8 showed frequent mutations, while envelop, membrane, ORF6, ORF7a and ORF7b showed conservation in terms of amino acid substitutions. Combined analysis with the frequently mutated residues identified 20 viral variants, among which 12 specific combinations comprised more than 97% of the isolates considered for the analysis. Some of the mutations across different proteins showed co-occurrences, suggesting their structural and/or functional interaction among different SARS-COV-2 proteins, and their involvement in adaptability and viral transmission. Analysis of protein structure stability of surface glycoprotein mutants indicated the viability of specific variants and are more prone to be temporally and spatially distributed across the globe. A similar empirical analysis of other proteins indicated the existence of important functional implications of several variants. Identification of frequently mutated variants among COVID-19 patients might be useful for better clinical management, contact tracing, and containment of the disease.

Inhibition of Plasmodium falciparum cysteine protease falcipain-2 by a human cross-class inhibitor serpinB3: A mechanistic insight.

Falcipain-2(FP2), a cysteine protease from Plasmodium falciparum, cleaves host erythrocyte hemoglobin and specific membrane skeleton components during the parasite life cycle. Therefore its inhibition has been considered as an attractive approach to combat the disease. SerpinB3 (SPB3) belongs to the ovalbumin-serpin family and is a potent cross-class inhibitor of cysteine cathepsins L, K, S and papain. This study explored the possibility of inhibition of FP2 by SPB3. It turned out that general proteolytic activities as well as specific hemoglobinolytic activity of FP2 have been inhibited by SPB3. Furthermore, studies have been designed to investigate and characterize the mechanism of inhibition in comparison with proteases Cathepsin L (CTSL) and papain. The Ki value of inhibition for FP2, measured against its specific substrate (VLK-pNA), is 338.11 nM and stoichiometry (I/E ratio) of inhibition is 1. These values are comparable to CTSL and papain. Analytical gel filtration profile and CD spectroscopy data confirm FP2-SPB3 complex formation. Our studies revealed that interaction of SPB3 with FP2 is non-covalent type like that of CTSL and papain but unlike other serine protease-inhibiting serpins. An in-silico docking and simulation study have been performed with FP2 as well as CTSL and results suggest different binding mode for FP2 and CTSL, though both the complexes are stable with significant contribution from electrostatic energy of interaction. We further showed a disease state mutant SPB3-Gly351Ala performed better anti-protease activity against FP2. This study, for the first time, has shown a serpin family inhibitor from human could efficiently inhibit activity of FP2.

Exploring protein-protein intermolecular recognition between meprin-α and endogenous protease regulator cystatinC coupled with pharmacophore elucidation.

Meprins are a group of zinc metalloproteases of the astacin family which play a pivotal role in several physiological and pathologocal diseases. The inhibition of the meprins by various inhibitors, macromolecular and small molecules, is crucial in the control of several diseases. Human cystatinC, an amyloidogenic protein, is reported to be an endogenous inhibitor of meprin-α. In this computational study, we elucidate a rational model for meprinα-cystatinC complex using protein-protein docking. The complex model as well as the unbound form was evaluated by molecular dynamics simulation. A simulation study revealed higher stability of the complex owing to the presence of several interactions. Virtual alanine mutagenesis helps in identifying the hotspots on both proteins. Based on the frequency of occurrence of hotspot amino acids, it was possible to enumerate the important amino acids primarily responsible for protein stability present at the amino-terminal end of cystatin. Finally, pharmacophore elucidation carried out based on the information obtained from a series of small molecular inhibitors against meprin-α can be utilized in future for rational drug design and therapy.

Not all pycnodysostosis-related mutants of human cathepsin K are inactive - crystal structure and biochemical studies of an active mutant I249T.

Human cathepsin K (CTSK) is a collagenolytic lysosomal cysteine protease that plays an important role in bone turnover. Mutation in CTSK gene is associated with loss of collagenolytic activity of CTSK leading to an autosomal recessive bone disorder called pycnodysostosis. Although a number of pycnodysostotic missense mutations have been reported, underlying mechanism of the disease is not clear. In this study, we investigated in vitro six recombinant pycnodysostosis-related mutants of human CTSK (G79E, I249T, G243E, G303E, G319C and Q187P). While all the mutants, like wild-type, show similar high levels of expression in Escherichia coli, four of them (G79E, G303E, G319C and Q187P) are inactive, unstable and spontaneously degrade during purification process. In contrast, proteolytic/collagenolytic activity, zymogen activation kinetics and stability of G243E and I249T mutants are nominally affected. Crystal structure of I249T at 1.92 Å resolution shows that the mutation in R-domain causes conformational changes of a surface loop in the L-domain although the catalytic cleft remains unaltered. Molecular simulation, normal mode analysis and fluorescence lifetime measurement eliminated the possibility that the change in L-domain surface loop orientation is a crystallization artefact. CD-based thermal melting profile indicates that stability of I249T is significantly higher than wild-type. Our studies first time reports that pycnodysostosis-related mutations do not always lead to complete loss of general proteolytic activity or specific collagenolytic activity of CTSK. The first crystal structure of a pycnodysostotic mutant (I249T) provides critical information that may pave new avenues towards understanding the disease at molecular level. DATABASE: The atomic co-ordinates and structure factors for I249T mutant of human CTSK (codes 5Z5O) have been deposited in the Protein Data Bank (http://wwpdb.org/).

Highly Conserved Arg Residue of ERFNIN Motif of Pro-Domain is Important for pH-Induced Zymogen Activation Process in Cysteine Cathepsins K and L.

Pro-domain of a cysteine cathepsin contains a highly conserved Ex2Rx2Fx2Nx3Ix3N (ERFNIN) motif. The zymogen structure of cathepsins revealed that the Arg(R) residue of the motif is a central residue of a salt-bridge/H-bond network, stabilizing the scaffold of the pro-domain. Importance of the arginine is also demonstrated in studies where a single mutation (Arg → Trp) in human lysosomal cathepsin K (hCTSK) is linked to a bone-related genetic disorder "Pycnodysostosis". In the present study, we have characterized in vitro Arg → Trp mutant of hCTSK and the same mutant of hCTSL. The R → W mutant of hCTSK revealed that this mutation leads to an unstable zymogen that is spontaneously activated and auto-proteolytically degraded rapidly. In contrast, the same mutant of hCTSL is sufficiently stable and has proteolytic activity almost like its wild-type counterpart; however it shows an altered zymogen activation condition in terms of pH, temperature and time. Far and near UV circular dichroism and intrinsic tryptophan fluorescence experiments have revealed that the mutation has minimal effect on structure of the protease hCTSL. Molecular modeling studies shows that the mutated Trp31 in hCTSL forms an aromatic cluster with Tyr23 and Trp30 leading to a local stabilization of pro-domain and supplements the loss of salt-bridge interaction mediated by Arg31 in wild-type. In hCTSK-R31W mutant, due to presence of a non-aromatic Ser30 residue such interaction is not possible and may be responsible for local instability. These differences may cause detrimental effects of R31W mutation on the regulation of hCTSK auto-activation process compared to altered activation process in hCTSL.

Mutation in the Pro-Peptide Region of a Cysteine Protease Leads to Altered Activity and Specificity-A Structural and Biochemical Approach.

Papain-like proteases contain an N-terminal pro-peptide in their zymogen form that is important for correct folding and spatio-temporal regulation of the proteolytic activity of these proteases. Catalytic removal of the pro-peptide is required for the protease to become active. In this study, we have generated three different mutants of papain (I86F, I86L and I86A) by replacing the residue I86 in its pro-peptide region, which blocks the specificity determining S2-subsite of the catalytic cleft of the protease in its zymogen form with a view to investigate the effect of mutation on the catalytic activity of the protease. Steady-state enzyme kinetic analyses of the corresponding mutant proteases with specific peptide substrates show significant alteration of substrate specificity-I86F and I86L have 2.7 and 29.1 times higher kcat/Km values compared to the wild-type against substrates having Phe and Leu at P2 position, respectively, while I86A shows lower catalytic activity against majority of the substrates tested. Far-UV CD scan and molecular mass analyses of the mature form of the mutant proteases reveal similar CD spectra and intact masses to that of the wild-type. Crystal structures of zymogens of I86F and I86L mutants suggest that subtle reorganization of active site residues, including water, upon binding of the pro-peptide may allow the enzyme to achieve discriminatory substrate selectivity and catalytic efficiency. However, accurate and reliable predictions on alteration of substrate specificity require atomic resolution structure of the catalytic domain after zymogen activation, which remains a challenging task. In this study we demonstrate that through single amino acid substitution in pro-peptide, it is possible to modify the substrate specificity of papain and hence the pro-peptide of a protease can also be a useful target for altering its catalytic activity/specificity.

Ni(II)-Schiff base complex as an enzyme inhibitor of hen egg white lysozyme: a crystallographic and spectroscopic study.

The engineering of protein-small molecule interactions becomes imperative today to recognize the essential biochemical processes in living systems. Here we have investigated the interaction between hen egg white lysozyme (HEWL) and a newly synthesized small, simple nickel Schiff base complex (NSC) {(N(1)E,N(2)E)-N(1),N(2)-bis(pyridine-2-ylmethylene)propane-1,2-diaminenickel(II)} using different spectroscopic techniques. We attempted to determine the exact site of the interaction by crystallography. Absorption spectroscopy reveals that the interaction occurs through the ground state. The complex can quench the intrinsic fluorescence of HEWL through a static quenching method. The fluorescence quenching study along with the determination of thermodynamic parameters reveal that NSC binds HEWL spontaneously with moderate binding affinity. The results have also identified that the spontaneity of this enthalpy guided interaction is mainly governed by some H-bonding and hydrophobic interactions which are also indicated by the crystallographic analyses. Moreover, the crystallographic study shows that NSC makes its way into the active site enzyme cavity of HEWL forming a single covalent adduct between Ni(2+) and the oxygen of the active site Asp 52. The possibility of inhibiting the catalytic activity of HEWL by inclusion of NSC in the enzyme active site observed from crystallographic analyses has also been confirmed by enzyme kinetics experiments.

Enhancement of proteolytic activity of a thermostable papain-like protease by structure-based rational design.

Ervatamins (A, B and C) are papain-like cysteine proteases from the plant Ervatamia coronaria. Among Ervatamins, Ervatamin-C is a thermostable protease, but it shows lower catalytic efficiency. In contrast, Ervatamin-A which has a high amino acid sequence identity (∼90%) and structural homology (Cα rmsd 0.4 Å) with Ervatamin-C, has much higher catalytic efficiency (∼57 times). From the structural comparison of Ervatamin-A and -C, two residues Thr32 and Tyr67 in the catalytic cleft of Ervatamin-A have been identified whose contributions for higher activity of Ervatamin-A are established in our earlier studies. In this study, these two residues have been introduced in Ervatamin-C by site directed mutagenesis to enhance the catalytic efficiency of the thermostable protease. Two single mutants (S32T and A67Y) and one double mutant (S32T/A67Y) of Ervatamin-C have been generated and characterized. All the three mutants show ∼ 8 times higher catalytic efficiency (k cat/K m) than the wild-type. The thermostability of all the three mutant enzymes remained unchanged. The double mutant does not achieve the catalytic efficiency of the template enzyme Ervatamin-A. By modeling the structure of the double mutant and probing the role of active site residues by docking a substrate, the mechanistic insights of higher activity of the mutant protease have been addressed. The in-silico study demonstrates that the residues beyond the catalytic cleft also influence the substrate binding and positioning of the substrate at the catalytic centre, thus controlling the catalytic efficiency of an enzyme.

Protein interactions of Merocyanine 540: spectroscopic and crystallographic studies with lysozyme as a model protein.

Spectroscopic and crystallographic studies reveal that Merocyanine 540 (MC 540), a well-known therapeutically important anionic cyanine dye, interacts with hen egg white lysozyme in ground state. The formation of the complex is validated by two isosbestic points in absorption spectra of lysozyme with varied concentration of MC 540 and appearance of an isodichroic point in induced CD spectra of MC 540 with lysozyme. The blue shift of fluorescence maximum of lysozyme in presence of MC 540 shows hydrophobic effect on Trp due to complex formation probably through cooperative binding. Above 1:3M stoichiometric ratio (lysozyme:MC 540) an additional fluorescence hump arises because of structural changes in protein, where MC 540 acts as self-denaturant, inducing non-linearity in Stern-Volmer plot. The van't Hoff isotherms with negative changes in enthalpy at lower concentration and positive changes in entropy for entire concentration range of MC 540 depict the binding forces as hydrogen bonding/van der Waal's and ionic/hydrophobic respectively. Finally X-ray crystallographic structure of the complex shows that MC 540 adopts two conformations, cis and trans, while it binds to lysozyme. Benzoxole moiety of MC 540 interacts with Trp123 through π-stacking and SO3(2-) group is stabilized by ionic interaction/H-bonding with Arg125 of lysozyme.

The structure of a thermostable mutant of pro-papain reveals its activation mechanism.

Papain is the archetype of a broad class of cysteine proteases (clan C1A) that contain a pro-peptide in the zymogen form which is required for correct folding and spatio-temporal regulation of proteolytic activity in the initial stages after expression. This study reports the X-ray structure of the zymogen of a thermostable mutant of papain at 2.6 Å resolution. The overall structure, in particular that of the mature part of the protease, is similar to those of other members of the family. The structure provides an explanation for the molecular basis of the maintenance of latency of the proteolytic activity of the zymogen by its pro-segment at neutral pH. The structural analysis, together with biochemical and biophysical studies, demonstrated that the pro-segment of the zymogen undergoes a rearrangement in the form of a structural loosening at acidic pH which triggers the proteolytic activation cascade. This study further explains the bimolecular stepwise autocatalytic activation mechanism by limited proteolysis of the zymogen of papain at the molecular level. The possible factors responsible for the higher thermal stability of the papain mutant have also been analyzed.

C-Terminal extension of a plant cysteine protease modulates proteolytic activity through a partial inhibitory mechanism.

The amino acid sequence of ervatamin-C, a thermostable cysteine protease from a tropical plant, revealed an additional 24-amino-acid extension at its C-terminus (CT). The role of this extension peptide in zymogen activation, catalytic activity, folding and stability of the protease is reported. For this study, we expressed two recombinant forms of the protease in Escherichia coli, one retaining the CT-extension and the other with it truncated. The enzyme with the extension shows autocatalytic zymogen activation at a higher pH of 8.0, whereas deletion of the extension results in a more active form of the enzyme. This CT-extension was not found to be cleaved during autocatalysis or by limited proteolysis by different external proteases. Molecular modeling and simulation studies revealed that the CT-extension blocks some of the substrate-binding unprimed subsites including the specificity-determining subsite (S2) of the enzyme and thereby partially occludes accessibility of the substrates to the active site, which also corroborates the experimental observations. The CT-extension in the model structure shows tight packing with the catalytic domain of the enzyme, mediated by strong hydrophobic and H-bond interactions, thus restricting accessibility of its cleavage sites to the protease itself or to the external proteases. Kinetic stability analyses (T(50) and t(1/2) ) and refolding experiments show similar thermal stability and refolding efficiency for both forms. These data suggest that the CT-extension has an inhibitory role in the proteolytic activity of ervatamin-C but does not have a major role either in stabilizing the enzyme or in its folding mechanism.

Crystallization and preliminary X-ray diffraction studies of the precursor protein of a thermostable variant of papain.

The crystallization of a recombinant thermostable variant of pro-papain has been carried out. The mutant pro-enzyme was expressed in Escherichia coli as inclusion bodies, refolded, purified and crystallized. The crystals belonged to space group P2(1), with unit-cell parameters a = 42.9, b = 74.8, c = 116.5 Å, ß = 93.0°, and diffracted to 2.6 Å resolution using synchrotron radiation. Assuming the presence of two molecules in the asymmetric unit, the calculated Matthews coefficient is 2.28 Å(3) Da(-1), corresponding to a solvent content of 46%. Initial attempts to solve the structure using molecular-replacement techniques were successful.

Improving thermostability of papain through structure-based protein engineering.

Papain is a plant cysteine protease of industrial importance having a two-domain structure with its catalytic cleft located at the domain interface. A structure-based rational design approach has been used to improve the thermostability of papain, without perturbing its enzymatic activity, by introducing three mutations at its interdomain region. A thermostable homologue in papain family, Ervatamin C, has been used as a template for this purpose. A single (K174R), a double (K174RV32S) and a triple (K174RV32SG36S) mutant of papain have been generated, of which the triple mutant shows maximum thermostability with the half-life (t(1/2)) extended by 94 min at 60 degrees C and 45 min at 65 degrees C compared to the wild type (WT). The temperature of maximum enzymatic activity (T(max)) and 50% maximal activity (T(50)) for the triple mutant increased by 15 and 4 degrees C, respectively. Moreover, the triple mutant exhibits a faster inactivation rate beyond T(max) which may be a desirable feature for an industrial enzyme. The values of t(1/2) and T(max) for the double mutant lie between those of the WT and the triple mutant. The single mutant however turns out to be unstable for biochemical characterization. These results have been substantiated by molecular modeling studies which also indicate highest stability for the triple mutant based on higher number of interdomain H-bonds/salt-bridges, less interdomain flexibility and lower stability free-energy compared to the WT. In silico studies also explain the unstable behavior of the single mutant.

Production and recovery of recombinant propapain with high yield.

Papain (EC 3.4.22.2), the archetypal cysteine protease of C1 family, is of considerable commercial significance. In order to obtain substantial quantities of active papain, the DNA coding for propapain, the papain precursor, has been cloned and expressed at a high level in Escherichia coli BL21(DE3) transformed with two T7 promoter based pET expression vectors - pET30 Ek/LIC and pET28a(+) each containing the propapain gene. In both cases, recombinant propapain was expressed as an insoluble His-tagged fusion protein, which was solubilized, and purified by nickel chelation affinity chromatography under denaturing conditions. By systematic variation of parameters influencing the folding, disulfide bond formation and prevention of aggregate formation, a straightforward refolding procedure, based on dilution method, has been designed. This refolded protein was subjected to size exclusion chromatography to remove impurities and around 400mg of properly refolded propapain was obtained from 1L of bacterial culture. The expressed protein was further verified by Western blot analysis by cross-reacting it with a polyclonal anti-papain antibody and the proteolytic activity was confirmed by gelatin SDS-PAGE. This refolded propapain could be converted to mature active papain by autocatalytic processing at low pH and the recombinant papain so obtained has a specific activity closely similar to the native papain. This is a simple and efficient expression and purification procedure to obtain a yield of active papain, which is the highest reported so far for any recombinant plant cysteine protease.

Structural insights into the substrate specificity and activity of ervatamins, the papain-like cysteine proteases from a tropical plant, Ervatamia coronaria.

Multiple proteases of the same family are quite often present in the same species in biological systems. These multiple proteases, despite having high homology in their primary and tertiary structures, show deviations in properties such as stability, activity, and specificity. It is of interest, therefore, to compare the structures of these multiple proteases in a single species to identify the structural changes, if any, that may be responsible for such deviations. Ervatamin-A, ervatamin-B and ervatamin-C are three such papain-like cysteine proteases found in the latex of the tropical plant Ervatamia coronaria, and are known not only for their high stability over a wide range of temperature and pH, but also for variations in activity and specificity among themselves and among other members of the family. Here we report the crystal structures of ervatamin-A and ervatamin-C, complexed with an irreversible inhibitor 1-[l-N-(trans-epoxysuccinyl)leucyl]amino-4-guanidinobutane (E-64), together with enzyme kinetics and molecular dynamic simulation studies. A comparison of these results with the earlier structures helps in a correlation of the structural features with the corresponding functional properties. The specificity constants (k(cat)/K(m)) for the ervatamins indicate that all of these enzymes have specificity for a branched hydrophobic residue at the P2 position of the peptide substrates, with different degrees of efficiency. A single amino acid change, as compared to ervatamin-C, in the S2 pocket of ervatamin-A (Ala67-->Tyr) results in a 57-fold increase in its k(cat)/K(m) value for a substrate having a Val at the P2 position. Our studies indicate a higher enzymatic activity of ervatamin-A, which has been subsequently explained at the molecular level from the three-dimensional structure of the enzyme and in the context of its helix polarizibility and active site plasticity.

A thermostable cysteine protease precursor from a tropical plant contains an unusual C-terminal propeptide: cDNA cloning, sequence comparison and molecular modeling studies.

We report here the cloning and characterization of the entire cDNA of a papain-like cysteine protease from a tropical flowering plant. The 1098-bp ORF of the cDNA codify a protease precursor having a signal peptide of 19 amino acids, a cathepsin-L like N-terminal proregion of 114 amino acids, a mature enzyme part of 208 amino acids and a C-terminal proregion of 24 amino acids. The derived amino acid sequence of the mature part tallies with the thermostable cysteine protease Ervatamin-C--as was aimed at. The C-terminal proregion of the protease has altogether a different sequence pattern not observed in other members of the family and it contains a negatively charged helical zone. The three-dimensional model of the precursor, based on the homology modeling and X-ray structure, shows that the extended peptide stretch region of the N-terminal propeptide, covering the interdomain cleft, contains protruding side chains of positively charged residues. This study also indicates that the negatively charged zone of C-terminal propeptide may interact with the positively charged zone of the N-terminal propeptide in a cooperative manner in the maturation process of this enzyme.

Crystallization and preliminary X-ray diffraction studies of the cysteine protease ervatamin A from Ervatamia coronaria.

The ervatamins are highly stable cysteine proteases that are present in the latex of the medicinal plant Ervatamia coronaria and belong to the papain family, members of which share similar amino-acid sequences and also a similar fold comprising two domains. Ervatamin A from this family, a highly active protease compared with others from the same source, has been purified to homogeneity by ion-exchange chromatography and crystallized by the vapour-diffusion method. Needle-shaped crystals of ervatamin A diffract to 2.1 A resolution and belong to space group C222(1), with unit-cell parameters a = 31.10, b = 144.17, c = 108.61 A. The solvent content using an ervatamin A molecular weight of 27.6 kDa is 43.9%, with a VM value of 2.19 A3 Da(-1) assuming one protein molecule in the asymmetric unit. A molecular-replacement solution has been found using the structure of ervatamin C as a search model.