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.

Role of remote scaffolding residues in the inhibitory loop pre-organization, flexibility, rigidification and enzyme inhibition of serine protease inhibitors.

Canonical serine protease inhibitors interact with cognate enzymes through the P3-P2' region of the inhibitory loop while its scaffold hardly makes any contact. Neighboring scaffolding residues like Arginines or Asparagine shape-up the inhibitory loop and favor the resynthesis of cleaved scissile bond. However, role of remote scaffolding residues, which are not involved in religation, was not properly explored. Crystal structures of two engineered winged bean chymotrypsin inhibitor (WCI) complexed with Bovine trypsin (BPT) namely L65R-WCI:BPT and F64Y/L65R-WCI:BPT show that the inhibitory loop of these engineered inhibitors are recognized and rigidified properly at the enzyme active site like other strong trypsin inhibitors. Chimeric protein ETI(L)-WCI(S), having a loop of Erythrina caffra Trypsin Inhibitor, ETI on the scaffold of WCI, was previously shown to behave like substrate. Non-canonical structure of the inhibitory loop and its flexibility are attributed to the presence of smaller scaffolding residues which cannot act as barrier to the inhibitory loop like in ETI. Double mutant A76R/L115Y-(ETI(L)-WCI(S)), where the barrier is reintroduced on ETI(L)-WCI(S), shows regaining of inhibitory activity. The structure of A76R/L115Y-(ETI(L)-WCI(S)) along with L65R-WCI:BPT and F64Y/L65R-WCI:BPT demonstrate here that the lost canonical conformation of the inhibitory loop is fully restored and loop flexibility is dramatically reduced. Therefore, residues at the inhibitory loop interact with the enzyme playing the primary role in recognition and binding but scaffolding residues having no direct interaction with the enzyme are crucial for rigidification event and the inhibitory potency. B-factor analysis indicates that the amount of inhibitory loop rigidification varies between different inhibitor families.

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.

Identification of a novel set of scaffolding residues that are instrumental for the inhibitory property of Kunitz (STI) inhibitors.

For canonical serine protease inhibitors (SPIs), scaffolding spacer residue Asn or Arg religates cleaved scissile peptide bond to offer efficient inhibition. However, several designed "mini-proteins," containing the inhibitory loop and the spacer(s) with trimmed scaffold behave like substrates, indicating that scaffolding region beyond the spacer is also important in the inhibitory process. To understand the loop-scaffold compatibility, we prepared three chimeric proteins ECI(L)-WCI(S), ETI(L)-WCI(S), and STI(L)-WCI(S), where the inhibitory loop of ECI, ETI, and STI is placed on the scaffold of their homolog WCI. Results show that although ECI(L)-WCI(S) and STI(L)-WCI(S) behave like good inhibitors, ETI(L)-WCI(S) behaves like a substrate. That means a set of loop residues (SRLRSAFI), offering strong trypsin inhibition in ETI, act as a substrate when they seat on the scaffold of WCI. Crystal structure of ETI(L)-WCI(S) shows that the inhibitory loop is of noncanonical conformation. We identified three novel scaffolding residues Trp88, Arg74, and Tyr113 in ETI that act as barrier to confine the inhibitory loop to canonical conformation. Absence of this barrier in the scaffold of WCI makes the inhibitory loop flexible in ETI(L)-WCI(S) leading to a loss of canonical conformation, explaining its substrate-like behavior. Incorporation of this barrier back in ETI(L)-WCI(S) through mutations increases its inhibitory power, supporting our proposition. Our study provides structural evidence for the contribution of remote scaffolding residues in the inhibitory process of canonical SPIs. Additionally, we rationalize why the loop-scaffold swapping is not permitted even among the members of highly homologous inhibitors, which might be important in the light of inhibitor design.

Structural determinants of V. cholerae CheYs that discriminate them in FliM binding: comparative modeling and MD simulation studies.

Chemotaxis of Vibrio cholerae is a complex process where multiple paralogues of various chemotaxis genes participate. V. cholerae contains five copies of the response regulator protein CheY (CheYV) and the role played by these CheY homologs in chemotaxis and virulence are investigated only through a few in vivo studies. As identification of the molecular features that discriminate CheYVs in terms of FliM binding is necessary for the detailed understanding of chemotaxis and pathogenesis, we built the models of CheYVs through comparative modeling and MD simulation was performed on each model in their phosphorylated and Mg+2 bound state. Our analysis identified the key structural elements, unique to CheY3V, which complement the N-terminal part of FliMV and we explained how the structure, shape, and surface properties of the FliM binding pocket of other CheYVs abrogate this function. Furthermore, we have provided the structural basis of a putative cross species interaction between CheYE and FliMV, identified in a recent in vivo study.

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.

Cloning, expression, purification, crystallization and preliminary crystallographic analysis of pseudo death-effector domain of HIPPI, a molecular partner of Huntingtin-interacting protein HIP-1.

The formation of a heterodimer between Huntingtin-interacting protein-1 (HIP-1) and its novel partner HIPPI (HIP-1 protein interactor) through their pseudo death-effector domains (pDEDs) is a key step that recruits caspase-8 and initiates apoptosis. This could be one of the pathways by which apoptosis is increased in Huntington's disease (HD). A construct consisting of the pDED of HIPPI has been cloned and overexpressed as 6NH-tagged protein and purified by Ni-NTA affinity chromatography. Crystals of the pDED of HIPPI were grown in space group P4(1), with unit-cell parameters a = b = 77.42, c = 33.31 A and a calculated Matthews coefficient of 1.88 A3 Da(-1) (33% solvent content) with two molecules per asymmetric unit.

Spacer Asn determines the fate of Kunitz (STI) inhibitors, as revealed by structural and biochemical studies on WCI mutants.

The scaffold of serine protease inhibitors plays a significant role in the process of religation which resists proteolysis of the inhibitor in comparison to a substrate. Although the role of the conserved scaffolding Asn residue was previously implicated in the maintenance of the binding loop conformation of Kunitz (STI) inhibitors, its possible involvement in the prevention of proteolysis is still unexplored. In this paper, we have investigated the specific role of the spacer Asn in the prevention of proteolysis through structural and biochemical studies on the mutants where Asn14 of winged bean chymotrypsin inhibitor (WCI) has been replaced by Gly, Ala, Thr, Leu, and Gln. A residue having no side chain or beta-branching at the 14th position creates deformation and insufficient protrusion of the binding loop, and as a result N14G and N14T lose the ability to recognize proteases. Although the reactive site loop conformation of N14A and N14Q are almost identical to WCI, biochemical results present N14A as a substrate indicating that the methyl group of Ala14 is not suitable to capture the cleaved parts together for religation. The poor inhibitory power of N14L points toward the chemical incompatibility of Leu at the 14th position, although its size is the same as Asn; on the other hand, slight loss of inhibitory potency of N14Q is attributed to the inappropriate placement of the Gln14 polar head, caused by the strained accommodation of its bigger side chain. These observations collectively allow us to conclude that the side chain of spacer Asn fits snugly into the concave space of the reactive site loop cavity and its ND2 atom forms hydrogen bonds with the P2 and P1' carbonyl O at either side of the scissile bond holding the cleaved products together for religation. Through database analysis, we have identified such spacer asparagines in five other families of serine protease inhibitors with a similar disposition of their ND2 atoms, which supports our proposition.

Purification, crystallization and preliminary X-ray structural studies of a 7.2 kDa cytotoxin isolated from the venom of Daboia russelli russelli of the Viperidae family.

A cytotoxin (MW 7.2 kDa) from Indian Russell's viper (Daboia russelli russelli) venom possessing antiproliferative activity, cardiotoxicity, neurotoxicity and myotoxicity has been purified, characterized and crystallized. The crystals belong to the tetragonal space group P4(1), with unit-cell parameters a = b = 47.94, c = 50.2 A. Larger crystals, which diffracted to 1.5 A, were found to be twinned; diffraction data were therefore collected to 2.93 A resolution using a smaller crystal. Molecular-replacement calculations identified two molecules of the protein in the asymmetric unit, which is in accordance with the calculated VM value.

Single mutation at P1 of a chymotrypsin inhibitor changes it to a trypsin inhibitor: X-ray structural (2.15 A) and biochemical basis.

Change in specificity, caused by the mutations at P1 site, of the serine protease inhibitors of different families is reported in the literature, but Kunitz (STI) family inhibitors are almost unexplored in this regard. In this paper, we present the crystal structure of a P1 variant of winged bean chymotrypsin inhibitor (WCI) belonging to Kunitz (STI) family, supplemented by biochemical, phylogenetic and docking studies on the mutant. A single mutation (Leu-->Arg) at P1 converted WCI to a strong inhibitor of trypsin with an association constant of 4.8x10(10) M(-1) which is comparable to other potent trypsin inhibitors of the family. The crystal structure (2.15 A) of this mutant (L65R) shows that its reactive site loop conformation deviates from that of WCI and adopts a structure similar to that of Erythrina caffra trypsin inhibitor (ETI) belonging to the same family. Mutation induced structural changes have also been propagated in a concerted manner to the neighboring conserved scaffolding residue Asn14, such that the side chain of this residue took an orientation similar to that of ETI and optimized the hydrogen bonds with the loop residues. While docking studies provide information about the accommodation of non-specific residues in the active site groove of trypsin, the basis of the directional alteration of the reactive site loop conformation has been understood through sequence analysis and related phylogenetic studies.

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.

Crystal structures of HbA2 and HbE and modeling of hemoglobin delta 4: interpretation of the thermal stability and the antisickling effect of HbA2 and identification of the ferrocyanide binding site in Hb.

Hemoglobin A(2) (alpha(2)delta(2)) is an important hemoglobin variant which is a minor component (2-3%) in the circulating red blood cells, and its elevated concentration in beta-thalassemia is a useful clinical diagnostic. In beta-thalassemia major, where there is beta-chain production failure, HbA(2) acts as the predominant oxygen deliverer. HbA(2) has two more important features. (1) It is more resistant to thermal denaturation than HbA, and (2) it inhibits the polymerization of deoxy sickle hemoglobin (HbS). Hemoglobin E (E26K(beta)), formed as a result of the splice site mutation on exon 1 of the beta-globin gene, is another important hemoglobin variant which is known to be unstable at high temperatures. Both heterozygous HbE (HbAE) and homozygous HbE (HbEE) are benign disorders, but when HbE combines with beta-thalassemia, it causes E/beta-thalassemia which has severe clinical consequences. In this paper, we present the crystal structures of HbA(2) and HbE at 2.20 and 1.74 A resolution, respectively, in their R2 states, which have been used here to provide the probable explanations of the thermal stability and instability of HbA(2) and HbE. Using the coordinates of R2 state HbA(2), we modeled the structure of T state HbA(2) which allowed us to address the structural basis of the antisickling property of HbA(2). Using the coordinates of the delta-chain of HbA(2) (R2 state), we also modeled the structure of hemoglobin homotetramer delta(4) that occurs in the case of rare HbH disease. From the differences in intersubunit contacts among beta(4), gamma(4), and delta(4), we formed a hypothesis regarding the possible tetramerization pathway of delta(4). The crystal structure of a ferrocyanide-bound HbA(2) at 1.88 A resolution is also presented here, which throws light on the location and the mode of binding of ferrocyanide anion with hemoglobin, predominantly using the residues involved in DPG binding. The pH dependence of ferrocyanide binding with hemoglobin has also been investigated.

Structural basis of the unusual stability and substrate specificity of ervatamin C, a plant cysteine protease from Ervatamia coronaria.

Ervatamin C is an unusually stable cysteine protease from the medicinal plant Ervatamia coronaria belonging to the papain family. Though it cleaves denatured natural proteins with high specific activity, its activity toward some small synthetic substrates is found to be insignificant. The three-dimensional structure and amino acid sequence of the protein have been determined from X-ray diffraction data at 1.9 A (R = 17.7% and R(free) = 19.0%). The overall structure of ervatamin C is similar to those of other homologous cysteine proteases of the family, folding into two distinct left and right domains separated by an active site cleft. However, substitution of a few amino acid residues, which are conserved in the other members of the family, has been observed in both the domains and also at the region of the interdomain cleft. Consequently, the number of intra- and interdomain hydrogen-bonding interactions is enhanced in the structure of ervatamin C. Moreover, a unique disulfide bond has been identified in the right domain of the structure, in addition to the three conserved disulfide bridges present in the papain family. All these factors contribute to an increase in the stability of ervatamin C. In this enzyme, the nature of the S2 subsite, which is the primary determinant of specificity of these proteases, is similar to that of papain, but at the S3 subsite, Ala67 replaces an aromatic residue, and has the effect of eliminating sufficient hydrophobic interactions required for S3-P3 stabilization. This provides the possible explanation for the lower activity of ervatamin C toward the small substrate/inhibitor. This substitution, however, does not affect the binding of denatured natural protein substrates to the enzyme significantly, as there exist a number of additional interactions at the enzyme-substrate interface outside the active site cleft.

Proposed amino acid sequence and the 1.63 A X-ray crystal structure of a plant cysteine protease, ervatamin B: some insights into the structural basis of its stability and substrate specificity.

The crystal structure of a cysteine protease ervatamin B, isolated from the medicinal plant Ervatamia coronaria, has been determined at 1.63 A. The unknown primary structure of the enzyme could also be traced from the high-quality electron density map. The final refined model, consisting of 215 amino acid residues, 208 water molecules, and a thiosulfate ligand molecule, has a crystallographic R-factor of 15.9% and a free R-factor of 18.2% for F > 2sigma(F). The protein belongs to the papain superfamily of cysteine proteases and has some unique properties compared to other members of the family. Though the overall fold of the structure, comprising two domains, is similar to the others, a few natural substitutions of conserved amino acid residues at the interdomain cleft of ervatamin B are expected to increase the stability of the protein. The substitution of a lysine residue by an arginine (residue 177) in this region of the protein may be important, because Lys --> Arg substitution is reported to increase the stability of proteins. Another substitution in this cleft region that helps to hold the domains together through hydrogen bonds is Ser36, replacing a conserved glycine residue in the others. There are also some substitutions in and around the active site cleft. Residues Tyr67, Pro68, Val157, and Ser205 in papain are replaced by Trp67, Met68, Gln156, and Leu208, respectively, in ervatamin B, which reduces the volume of the S2 subsite to almost one-fourth that of papain, and this in turn alters the substrate specificity of the enzyme.

Oxorhenium(V) and Oxotechnetium(V) Complexes of Cysteine.

Earlier attempts to obtain technetium complexes with cysteine always resulted in the formation of a product contaminated with polymeric species. A pure product, which could be chemically characterized and adopted for radiopharmaceutical preparation, has now been obtained by using cystine as the precursor of cysteine. This method has been extended to prepare the corresponding rhenium chelate, isolated as the tetraphenylphosphonium salt [Ph(4)P](+)[{ReO(Cys)(2)}(-){HReO(Cys)(2)}].4H(2)O. The X-ray crystal structure of this compound revealed the presence of both neutral and anionic chelated species. In [HReO(Cys)(2)], the cysteine carboxylate moiety is unidentatedly bound to rhenium, while the carboxylic acid of the second cysteine remains as free COOH. The coordination environment around rhenium in the anionic species [ ReO(Cys)(2)(-)] is similar, the only difference being that the uncoordinated carboxylate moiety is present as a COO(-) anion. The thiolate, amine coordination of the ligand with the metal is present in both the chelate units. The compound crystallized in an orthorhombic system with the space group P2(1)2(1)2(1), and having four formula units in each cell. The crystal data are a = 9.700(2) Å, b = 12.836(3) Å, and c = 36.228(3) Å. The rhenium chelate has been structurally correlated with the technetium chelates through comparable spectroscopic and chromatographic data. The technetium-99m analogue of this rhenium chelate exhibited renal tubular transport and renal retention, which makes this radiopharmaceutical useful for evaluation of the clinical status of renal patients.