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

Abstract

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.

Computational Study on Substrate Specificity of a Novel Cysteine Protease 1 Precursor from Zea mays

“…This result indicates that the enzyme has specificity for aromatic or non-polar residues (such as Phe and Tyr) at the P2 position of the peptide substrate. The crystal structure of zmCP1 complexes with different substrate analog inhibitors reveals that the P1 side chain faces the solvent, whereas the P2 side chain contacts the enzyme surface inside an enclosed cavity, the S2 subsites which in papain is dominantly hydrophobic in nature [27,28,29]. Our result is consistent with the existing data [21].…”

“…It was reported that the S2 pocket in C1A family proteases is of special interest, because it is an established fact that the specificity of this family enzymes is determined predominantly by P2–S2 interactions [13,27]. In our study, AMC was used as the fluorogenic group and Arg as the P1 residue.…”

Purification of a novel cysteine protease, procerain B, from Calotropis procera with distinct characteristics compared to procerain

“…The total cysteine content of procerain B is found to be nine (measured value 8.86) with one free cysteine residues (measured value 0.89), thus forming four disulphide bridges. Most of the cysteine proteases, including procerain, reported to have total seven cysteine, six cysteine forming three disulfides and one catalytic cysteine [25][26][27][28][29]. However, 11 cysteine [5 disulfide and 1 free] is also reported in plant cysteine protease, heynein [23].…”

Conserved water-mediated H-bonding dynamics of catalytic His159 and Asp158: insight into a possible acid–base coupled mechanism in plant thiol protease

“…Only one molecule was present in the asymmetric unit of the crystal structures of 1PPN [18], 9PAP [19], 1PE6 [20], 1PPP [21], 1KHP [22], 1KHQ [22], 1BP4 [23], 1AEC [24], 2ACT [25], 1MEG [26], 1YAL [27], 1GEC [28], and 1IWD [29], whereas two molecules were present in the asymmetric unit of 2PNS [30]. In the other two structures, 1CQD [31] and 2BDZ (RCSB database, 2005), the enzyme crystallized as a tetramer.…”