(The) structural and functional importance of a Ser-2 residue and a disulfide bond in serratia marcescens metalloprotease inhibitor

Abstract

Serratia marcescens metalloprotease inhibitor (SmaPI) is a proteinase inhibitor toward Serratia marcescens metalloprotease (SMP). The structure of SmaPI-SMP complex was not determined. However, the crystal structure of SMP-Erwinia chrysanthemi inhibitor was elucidated. The N-terminal sequence of E. chrysanthemi inhibitor is highly homologous to SmaPI. Therefore, the three dimensional structure of SmaPI-SMP complex was simulated by computer-modelling. Interestingly, the pocket around Ser-2 residue of SmaPI in the complex has enough space to be occupied by bulky side chain amino acids such as Phe and Tyr. To elucidate the roles of side chain of Ser-2 and the pocket in the complex, Seven point mutations at Ser-2 residue were introduced by site directed mutagenesis. Both of two mutants (S2K and S2D) showed very decreased inhibitory activities. A S2N mutant had a slightly decreased inhibitory activity. Four mutants (S2F, S2Y, S2A and S2V) had similar inhibitory activities compared with wild type. To investigate the role of a conserved disulfide bond in serralysin family inhibitors, we introduced two mutations (C24S and C47S) in SmaPI and expressed two mutant proteins in various versions of multiple-protease deficient B. subtilis DB431, WB600, and LB700. The substantial expression level of wild type SmaPI was maintained in the strains of B. subtilis, surprisingly, two mutants (C24S and C47S) were trivially expressed in lower versions of B.subtilis DB431 and WB600, however, substantially expressed in wprA-deficient B. subtilis LB700. In addition, the thermostability of two mutants (C24S and C47S) was monitored, the inhibitory activities of the mutants was dramatically decreased in boiling condition. In summary, N-terminal hydrophobicity of SmaPI is important in the inhibitory activity and a disulfide bond in SmaPI contributes to not only its thermostability but also protease-resistant conformation.