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 for a long time poorly understood, but recent work1,2,3 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 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 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.

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Figure 1: Biological role of RodA and evolutionary co-variation fold prediction.
Figure 2: Structure of RodA.
Figure 3: The central cavity is essential for RodA function.
Figure 4: Interaction between RodA and its class B pencillin-binding protein, PBP2.

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Acknowledgements

Financial support for the work was provided by NIH grant U19AI109764 (A.C.K., D.Z.R., T.G.B., S.W. and D. K.), NIH grant R01GM106303 (D.S.M.) and a CIHR doctoral research award to P.D.A.R. We thank Advanced Photon Source GM/CA beamline staff for technical support during X-ray data collection, K. Arnett (Harvard Center for Macromolecular Interactions) for support of circular dichroism experiments and C. Sander for discussions.

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Contributions

M.S. and A.J.M. performed expression screening experiments, and M.S. performed large-scale purification and crystallization of RodA as well as enzyme assays and circular dichroism spectroscopy. Additional input regarding enzyme assays was provided by P.D.A.R, V.S., D.K. and S.W. The structure was solved and refined by M.S. and A.C.K. using evolutionary coupling-derived models developed by K.B., A.G.G., T.A.H. and D.S.M. Assessment of RodA mutant phenotypes was conducted by G.D. and P.D.A.R. with supervision from T.G.B. and D.Z.R. Overall project supervision was performed by A.C.K. with input from T.G.B. and D.Z.R. The manuscript was written by M.S. and A.C.K. with input from other authors.

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Correspondence to Andrew C. Kruse.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks R. Read, K. Young and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Representative electron density.

ac, Simulated annealing composite omit 2Fo − Fc electron density map of T. thermophilus RodA contoured at 1.0σ within a 2.0 Å radius of atoms shown. d, The same map contoured at 1.0σ and coloured blue and green for RodA and water molecules, respectively. The modelled water molecules are shown as red spheres. e, The same map contoured at 1.0σ within a 3.0 Å radius RodA (shown in blue) and the same map contoured at 1.0σ for monoolein (shown in black).

Extended Data Figure 2 Comparison of RodA wild type and RodA(D255A) structures.

Structures of wild-type (green) and D255A (blue) RodA are shown viewed parallel to the membrane (left) and from the extracytoplasmic side (right). The Cα atom of residue 255 for each structure is shown as a sphere, and coloured pink (wild type) or yellow (D255A). The dashed lines represent the disordered residues 189–227 and 237–251 in both structures. The two structures are essentially identical, with a Cα r.m.s.d. of 0.1 Å.

Extended Data Figure 3 RodA sequence conservation.

The results of an alignment of 506 RodA sequences from diverse bacterial taxa, with representative examples displayed. Residues with 98%, 80% and 60% similarity across all 506 sequences are shown in black, grey and light grey, respectively. Secondary structure elements are shown above the alignment on the basis of the T. thermophilus RodA crystal structure and JPRED analysis of the portions of ECL4 that were not modelled in the structure.

Extended Data Figure 4 RodA evolutionary couplings.

a, The refined crystal structure of T. thermophilus RodA (shown in light blue) is in close agreement with its evolutionary couplings. Green, yellow and red lines between residues represent regions of the structure that are less than 5 Å, between 5 and 10 Å, and greater than 10 Å of the predicted evolutionary couplings, respectively. b, The partially disordered ECL4 in the refined structure is strongly coupled evolutionarily to the transmembrane domain of RodA. A representation of predicted intra- and inter-domain evolutionary couplings for ECL4 is mapped onto a EVfold model (shown in grey).

Extended Data Figure 5 Sequence conservation of T. thermophilus RodA.

a, Surface and ribbon representation of RodA (top and bottom, respectively). Analysis was performed using Consurf, coloured in a scale from teal (poorly conserved) to magenta (highly conserved). A bound lipid modelled as monoolein is shown in yellow sticks. The dashed lines represent disordered residues 189–227 and 237–251 in ECL4. b, The bound lipid (shown as spheres) is surrounded by many aliphatic amino acids (shown as sticks). c, Extracytoplasmic view of the water-filled central cavity and its proximity to the bound lipid molecule.

Extended Data Figure 6 Circular dichroism spectroscopy analysis of purified B. subtilis RodA variants.

Circular dichroism measurements of wild-type RodA as well as RodA(D280A), RodA(E117K/K120E) and RodA(I262S) indicate that the overall folds of all four forms are comparable and display characteristic α-helical peaks at 208 and 222 nm. For each panel, the circular dichroic spectra of wild-type and the indicated mutant RodA are shown in black and blue, respectively. The corresponding T. thermophilus numbering for each mutant is shown in parentheses. Experiments were repeated independently twice with similar results.

Extended Data Figure 7 Mutagenic analysis of B. subtilis RodA function in vivo.

Micrographs of B. subtilis strains harbouring an IPTG-inducible allele of wild-type rodA, and wild-type or mutant PxylA-rodA. Expression was induced by growing cells in the presence of 10 μM IPTG and 10 mM xylose. The bacterial cytosol is indicated by intracellular mCherry expression (Ppen-mCherry). Mutants in the central cavity show particularly deleterious phenotypes in this dominant negative assay. Experiments were repeated independently 2–3 times with similar results. A mutation made in the predicted RodA–bPBP interface does not prevent peptidoglycan polymerization in vitro (lower left panel), representative of two independent experiments.

Extended Data Figure 8 Evolutionary coupling analysis for the RodA–PBP2 complex.

Evolutionary co-variation maps highlighting 693 couplings for PBP2 (top left panel), and 362 couplings for RodA (bottom right panel) using a 95% confidence threshold. These maps display good correlation to the crystal structure of RodA and homology models of PBP2. The top right and lower bottom panels depict the 19 predicted inter-protein contacts between RodA and PBP2 using the same 95% confidence threshold.

Extended Data Table 1 Data collection and refinement statistics

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Sjodt, M., Brock, K., Dobihal, G. et al. Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis. Nature 556, 118–121 (2018). https://doi.org/10.1038/nature25985

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