Structural basis of peptidoglycan endopeptidase regulation

Abstract

<jats:title>Abstract</jats:title><jats:p>Most bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over, the latter process is mediated by PG cleavage enzymes, for example the endopeptidases (EPs). EPs themselves are essential for growth, but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (<jats:italic>e.g.</jats:italic>, β-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated <jats:italic>in vivo</jats:italic>, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogen <jats:italic>Vibrio cholerae.</jats:italic> Our data suggest that ShyA assumes two drastically different conformations; a more open form that allows for substrate binding, and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity <jats:italic>in vitro</jats:italic> and <jats:italic>in vivo</jats:italic>, and these results were recapitulated in EPs from the divergent pathogens <jats:italic>Neisseria gonorrheae</jats:italic> and <jats:italic>Escherichia coli</jats:italic>. Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain1 from the M23 active site, likely through conformational re-arrangement <jats:italic>in vivo</jats:italic>.</jats:p><jats:sec><jats:title>Significance</jats:title><jats:p>Bacteria digest their cell wall following exposure to antibiotics like penicillin. The endopeptidases (EPs) are among the proteins that catalyze cell wall digestion processes after antibiotic exposure, but we do not understand how these enzymes are regulated during normal growth. Herein, we present the structure of the major EP from the diarrheal pathogen <jats:italic>Vibrio cholerae.</jats:italic> Surprisingly, we find that EPs from this and other pathogens appear to be produced as a largely inactive precursor that undergoes a conformational shift exposing the active site to engage in cell wall digestion. These results enhance our understanding of how EPs are regulated and could open the door for the development of novel antibiotics that overactivate cell wall digestion processes.</jats:p></jats:sec>

Cell division is antagonized by the activity of peptidoglycan endopeptidases that promote cell elongation

“…In conclusion, our results indicate that PG endopeptidases can influence the division process in addition to promoting cell elongation, highlighting the critical role that controls governing endopeptidase activity play in the overall cell cycle. So far, the degradation of MepS by NlpI‐Prc and the presence of an inhibitory domain in MepM‐like endopeptidases are the only known factors affecting endopeptidase activity (Shin et al ., 2020). How these systems are modulated during growth or what signals they might respond to remain unclear.…”

The active repertoire of Escherichia coli peptidoglycan amidases varies with physiochemical environment

“…The EnvC LytM domain (residues 290‐419) was extracted using PyMOL (The PyMOL Molecular Graphics System, Version 1.8.4.0, Schrödinger, LLC) from the X‐ray structure downloaded from the RCSB PDB database (Berman et al., 2000) (Uniprot ID: P37690, residues 278‐419 PDB id: 4BH5 [Peters et al., 2013]). The 3D structures of NlpD (Uniprot ID: P0ADA3) and ActS (Uniprot ID: Q46798) were modeled using the Phyre web‐server (Kelley et al., 2015) using PDB id: 6U2A (Shin et al., 2020) as template for both structures. The LytM domains of NlpD (residues 261‐379) and ActS (residues 131‐248) were then extracted using PyMOL (The PyMOL Molecular Graphics System, Version 1.8.4.0, Schrödinger, LLC).…”

Understanding tolerance to cell wall–active antibiotics

“…70,[75][76][77][78] Since autolysin-mediated cleavage processes are potential threats to cellular structural integrity, they are likely carefully controlled or balanced by efficient cell wall synthesis at all times, and this has indeed been demonstrated for amidases and to some degree for EPs. 62,63,[79][80][81][82] Understanding the role and cleavage specificities of autolysins will be important to understand the mechanism of action of CWA antibiotics in the next sections.…”