Characterization of VldE (Spr1875), a Pneumococcal Two-State l,d-Endopeptidase with a Four-Zinc Cluster in the Active Site

Abstract

Remodeling of the pneumococcal cell wall, carried out by peptidoglycan (PG) hydrolases, is imperative for maintaining bacterial cell shape and ensuring survival, particularly during cell division or stress response. The Streptococcus pneumoniae protein Spr1875 plays a role in stress response, both regulated by the VicRK two-component system (analogous to the WalRK TCS found in Firmicutes). Modular Spr1875 presents a putative cell-wall binding module at the N-terminus and a catalytic C-terminal module (Spr1875^MT3) connected by a long linker. Assays of the full-length protein and Spr1875^MT3 with PG-based synthetic substrates by liquid chromatography/mass spectrometry revealed Spr1875 as an l,d-endopeptidase, renamed VldE (for VicRK-regulated l,d-endopeptidase), which hydrolyzed the cross-linked stem peptide in the PG. Remarkably, we observed asymmetric turnover with specific recognition of the acceptor peptide strand. Localization experiments showed that the protein is directed to the septum, which suggests that muralytic activity could be required for pneumococcal growth under stress conditions. Our findings, based on six high-resolution X-ray crystallographic structures and molecular-dynamics simulations, reveal two states for VldE^MT3. The protein transitions between a noncatalytic state that binds up to four zinc ions, thus behaving as a Zn²⁺ reservoir, and a catalytic state that performs the hydrolytic reaction with a single zinc ion. Furthermore, computational studies provide insight into the mechanism of catalytic-water activation and nucleophilic attack on the specific scissile peptide bond of the asymmetric cross-linked PG.