Molecular dynamics complemented by site-directed mutagenesis reveals significant difference between the interdomain...

Oligopeptidases B (OpdBs) are trypsin-like peptidases from protozoa and bacteria that belong to the prolyl oligopeptidase (POP) family. All POPs consist of C-terminal catalytic domain and N-terminal β-propeller domain and exist in two major conformations: closed (active), where the domains and residues of the catalytic triad are positioned close to each other, and open (non-active), where two domains and residues of the catalytic triad are separated. The interdomain interface, particularly, one of its salt bridges (SB1), plays a role in the transition between these two conformations. However, due to double amino acid substitution (E/R and R/Q), this functionally important SB1 is absent in γ-proteobacterial OpdBs including peptidase from Serratia proteamaculans (PSP). In this study, molecular dynamics was used to analyze inter- and intradomain interactions stabilizing PSP in the closed conformation, in which catalytic H652 is located close to other residues of the catalytic triad. The 3D models of either wild-type PSP or of mutant PSPs carrying activating mutations E125A and D649A in complexes with peptide-substrates were subjected to the analysis. The mechanism that regulates transition of H652 from active to non-active conformation upon domain separation in PSP and other γ-proteobacterial OpdB was proposed. The complex network of polar interactions within H652-loop/C-terminal α-helix and between these areas and β-propeller domain, established in silico, was in a good agreement with both previously published results on the effects of single-residue mutations and new data on the effects of the activating mutations on each other and on the low active mutant PSP-K655A.Communicated by Ramaswamy H. Sarma.