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

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.