Letter | Published:

Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis

Nature volume 556, pages 118121 (05 April 2018) | Download Citation

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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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.

Author information

Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Megan Sjodt
    •  & Andrew C. Kruse
  2. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Kelly Brock
    • , Anna G. Green
    • , Thomas A. Hopf
    •  & Debora S. Marks
  3. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Genevieve Dobihal
    • , Patricia D. A. Rohs
    • , Alexander J. Meeske
    • , Suzanne Walker
    • , Thomas G. Bernhardt
    •  & David Z. Rudner
  4. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA

    • Veerasak Srisuknimit
    •  & Daniel Kahne

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Andrew C. Kruse.

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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https://doi.org/10.1038/nature25985

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