Substrate binding and activation mechanism of the essential bacterial septal cell wall synthase FtsW

Abstract

Septal peptidoglycan (sPG) synthesis in most bacteria is driven by the essential, highly conserved FtsWIQLB synthase complex, comprising the SEDS (Shape, Elongation, Division, and Sporulation) glycosyltransferase FtsW, the monofunctional transpeptidase FtsI, and the scaffolding and regulatory subcomplex FtsQLB. Although apo structures of FtsWIQLB are known, no substrate-bound structures exist, likely due to the intrinsic flexibility of lipid II (L2) substrates. Here, we combined all-atom molecular dynamics simulations of E. coli FtsWIQLB with cysteine-based mutagenesis and cell-based assays to investigate donor and acceptor L2 binding sites in FtsW and to elucidate the complex's activation mechanism. We show that conserved arginine residues adjacent to membrane-accessible cavities in FtsW coordinate donor and acceptor L2 pyrophosphate groups, stabilizing substrate binding. The FtsWIQLB complex appears to adopt a self-inhibitory architecture in which gating elements and a periplasmic loop prevent the donor and acceptor from approaching FtsW's catalytic residue D297. Activation by FtsN or a superfission FtsW variant relieves these constraints, stabilizes acceptor-site binding, and reorients both substrates into a catalytically primed state. Long donor glycan chains further stabilize this activated conformation at the acceptor site, possibly promoting processive sPG polymerization. Comparative modeling of ESKAPE pathogen homologs reveals both conserved and divergent features of binding-site engagement and activation. We corroborate these computational findings with cysteine-based mutagenesis and crosslinking experiments. These results establish a mechanistic framework for FtsW substrate recognition and functional activation, highlighting tractable sites for structure- and mechanism-based antibiotic discovery targeting SEDS-class cell wall synthases.

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