Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis

The Shape, Elongation, Division, and Sporulation (“SEDS”) proteins are a large family of ubiquitous and essential transmembrane enzymes with critical roles in bacterial cell wall biology. The exact function of SEDS proteins was long enigmatic, but recent work(1–3) has revealed that the prototypical SEDS family member RodA is a peptidoglycan polymerase – a role previously attributed exclusively to members of the penicillin binding protein family(4). This discovery has made RodA and other SEDS proteins promising targets for the development of next-generation antibiotics. However, little is known regarding the molecular basis for SEDS activity, and no structural data are available for RodA or any homolog thereof. Here, we report the crystal structure of Thermus thermophilus RodA at a resolution of 2.9 Å, determined using evolutionary covariance-based fold prediction to enable molecular replacement. The structure reveals a novel ten-pass transmembrane fold with large extracellular loops, one of which is partially disordered. The protein contains a highly conserved cavity in the transmembrane domain, reminiscent of ligand binding sites in transmembrane receptors. Mutagenesis experiments in Bacillus subtilis and Escherichia coli show that perturbation of this cavity abolishes RodA function both in vitro and in vivo, indicating it is catalytically essential. These results provide a framework for understanding bacterial cell wall synthesis and SEDS protein function.