Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis

Evolutionary coupling-enabled molecular replacement determination of the structure of Thermus thermophilus RodA reveals a highly conserved cavity in its transmembrane domain, and mutagenesis experiments in Bacillus subtilis and Escherichia coli show that perturbation of this cavity abolishes RodA function. Structure of a new class of bacterial cell wall polymerases The SEDS (shape, elongation, division and sporulation) proteins are a large family of bacterial proteins important for cell wall synthesis. Following the discovery of a new family of peptidoglycan polymerases among the SEDS family, Andrew Kruse and colleagues report the first crystal structure of a member of this family, RodA. The team developed a new phasing methodology and carried out mutagenesis work that shows that RodA contains a ten-pass transmembrane fold. A highly conserved cavity in the transmembrane domain contains key residues and structural determinants that are important for RodA function. 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 for a long time poorly understood, but recent work 1 , 2 , 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 of SEDS activity, and no structural data are available for RodA or any homologue 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 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 that this cavity is catalytically essential. These results provide a framework for understanding bacterial cell wall synthesis and SEDS protein function.