Substrate binding and activation mechanism of the essential bacterial septal cell wall synthase FtsW
Septal peptidoglycan (sPG) synthesis in most bacteria is driven by the essential, highly conserved FtsWIOLB synthase complex, comprising the SEDS glycosyltransferase FtsW, the monofunctional transpeptidase FtsI, and the scaffolding subcomplex FtsOLB. Although apo structures of FtsWIOLB 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 FtsWIOLB with cysteine-based mutagenesis and cell-based assays to define donor and acceptor L2 binding sites in FtsW and to elucidate the complexs 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 FtsWIOLB complex appears to adopt a self-inhibitory architecture in which gating elements and a periplasmic loop prevent the donor and acceptor from approaching FtsWs 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, promoting processive sPG polymerization. Comparative modeling of ESKAPE pathogen homologs reveals 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.