A quantum mechanics/molecular mechanics study of the reaction mechanism of the hepatitis C virus NS3 protease with the NS5A/5B substrate

Combined quantum mechanics and molecular mechanics (QM/MM) calculations were carried out to characterize the reaction mechanism of the NS3 protease with its preferred substrate (NS5A/5B). The main purpose of this study was to locate the barrier states and intermediates along the distinguished coordinate path (DCP) involved in this process. These structures, and in particular the one corresponding to the first barrier state and intermediate (B1 and I1), could be a starting point for the synthesis of inhibitors of this protease, which could be used to treat hepatitis C. The two first steps of the reaction mechanism were studied, i.e., the acylation step and the breaking of the peptide bond. The first step takes place through a tetracoordinated intermediate, as suggested from previous works on other Serine proteases. The importance of the different amino acid residues was also considered (perturbation study where the MM charges of each residue were set to zero independently). The residues of the oxyanion hole were confirmed as the most important for the electrostatic stabilization of the tetracoordinate intermediate. Moreover, the role of other residues, e.g., Arg-155 and Asp-79, was also explained.     

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.