Structural Basis for the Essential Role of Ca²⁺ in the Lytic Activity of Staphylococcus aureus PlyGRCS Endolysin Targeting Methicillin‐Resistant Staphylococcus aureus

Abstract

Staphylococcus aureus causes a wide range of infections, from mild skin conditions to severe, life‐threatening diseases. Bacteriophage endolysins exhibit a selective capacity to degrade the peptidoglycan layer of Gram‐positive bacteria, making promising biotherapeutic agents against antibiotic‐resistant infections. PlyGRCS, a specific endolysin derived from S. aureus, comprises a catalytic CHAP domain and a cell‐wall binding SH3_5 domain connected by a linker. Ca2+ ions are essential for the CHAP domain's catalytic function. The crystal structure of PlyGRCS, determined in the absence of Ca2+ and refined to a resolution of 1.67 Å, revealed significant conformational changes in the Ca2+ binding site. Antimicrobial assays with Ca2+‐deficient PlyGRCS and mutants targeting key residues in the catalytic and Ca2+ binding regions highlighted the importance of specific functional residues for lytic activity against methicillin‐resistant Staphylococcus aureus (MRSA). These structural and microbial studies provide valuable insights into the critical residues contributing to PlyGRCS's bacteriolytic efficacy against MRSA.