Molecular Insights into Zn2+ Inhibition of the Antibacterial Endopeptidase Lysostaphin from Staphylococcus simulans

Abstract

Background: Mature lysostaphin (~28-kDa Lss) from Staphylococcus simulans proves effective in killing methicillin-resistant Staphylococcus aureus (MRSA) which is endemic in hospitals worldwide. Lss is Zn²⁺-dependent endopeptidase, but its bacteriolytic activity could be affected by exogenously added Zn²⁺.

Objective: To gain greater insights into structural and functional impacts of Zn²⁺and Ni²⁺on Lss-induced bioactivity.

Methods: Lss purified via immobilized metal ion-affinity chromatography was assessed for bioactivity using turbidity reduction assays. Conformational change of metal ion-treated Lss was examined by circular dichroism and intrinsic fluorescence spectroscopy. Co-sedimentation assay was performed to study interactions between Zn²⁺-treated Lss and S. aureus peptidoglycans. Metal ionbinding prediction and intermolecular docking were used to locate an extraneous Zn²⁺-binding site.

Results: A drastic decrease in Lss bioactivity against S. aureus and MRSA was revealed only when treated with Zn²⁺, but not Ni²⁺, albeit no negative effect of diethyldithiocarbamate-Zn²⁺-chelator on Lss-induced bioactivity. No severe conformational change was observed for Lss incubated with exogenous Zn²⁺ or Ni²⁺. Lss pre-treated with Zn²⁺ efficiently bound to S. aureus cell-wall peptidoglycans, suggesting non-interfering effect of exogenous metal ions on cell-wall targeting (CWT) activity. In silico analysis revealed that exogenous Zn²⁺, but not Ni²⁺, preferably interacted with a potential extraneous Zn²⁺-binding site (His253, Glu318 and His323) placed near the Zn²⁺-coordinating Lssactive site within the catalytic (CAT) domain.

Conclusion: Our present data signify the adverse influence of exogenous Zn²⁺ ions on Lss-induced staphylolytic activity through the exclusive presence within the CAT domain of an extraneous inhibitory Zn²⁺-binding site, without affecting the CWT activity.

Keywords: Inhibitory Zn2+-binding site; Zn2+-chelation; metallopeptidase; methicillin- resistant Staphylococcus aureus; peptidoglycan binding; staphylolytic activity.