Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis

Abstract

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 work1–3 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 family4. 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.

The EVcouplings Python framework for coevolutionary sequence analysis

“…ECs have been used successfully to predict the tertiary contacts (Marks, et al, 2011;Morcos, et al, 2011) and full 3D structure of proteins (Marks, et al, 2011) and RNA (Weinreb, et al, 2016), as well as protein-protein and protein-RNA interactions, disorder and conformational changes (Weigt, et al, 2009;Hopf, et al, 2012;Hopf, et al, 2014;Ovchinnikov, et al, 2014;Rodriguez-Rivas, et al, 2016;Toth-Petroczy, et al, 2016) and the effects of mutations (Figliuzzi, et al, 2015;Hopf, et al, 2017). More recently, hybrid approaches have integrated ECs with experimental data for improved structure determination using NMR, cryo-EM and X-ray crystallography data (Tang, et al, 2015;Sjodt, et al, 2018).…”

DHHC20 palmitoyl-transferase reshapes the membrane to foster catalysis

“…The ligands of many membrane-integral enzymes and transporters are hydrophobic (or at least amphipathic), implying these ligands are most likely recognized (and released) laterally, from (and to) the membrane instead of through the aqueous phase. In particular membraneintegral enzymes with lipid-like ligands include many transferases (20)(21)(22)(23)(24), as well as some translocases (25), synthases (26), polymerases (27) and proteases (28). Little is known about how most of these proteins interact with the membrane, but the increasing availability of structural data will surely help advance our understanding.…”

An activation pathway governs cell wall polymerization by a bacterial morphogenic machine

“…Therefore, following the discovery of PG polymerase activity for RodA, it was proposed that RodA-PBP2 and other SEDS-bPBP complexes form a functional PG synthase with both polymerase and crosslinking activity (Cho et al, 2016;Meeske et al, 2016). This possibility is supported by recent evolutionary coupling analyses and mutational studies indicating that a RodA-PBP2 complex formed through interactions between RodA and the pedestal domain of PBP2 is likely to be critical for Rod system function (Sjodt et al, 2018).…”

“…In both Escherichia coli and Bacillus subtilis, the aPBPs have been shown to display distinct subcellular localization dynamics from Rod system components and to be dispensable for the activity of the machinery (Cho et al, 2016;Meeske et al, 2016). It has therefore been proposed that a RodA-PBP2 complex forms the core PG synthase of the Rod system, an idea supported by recent evolutionary covariation analysis (Sjodt et al, 2018), and the finding that the aPBPs largely operate outside of the cytoskeletal system during cell elongation (Cho et al, 2016). A similar division of labor between aPBPs and FtsW-PBP3 may also be taking place during cytokinesis, but the relative contributions of the two types of synthases to the division process requires further definition.…”