Abstract
The shape, elongation, division and sporulation (SEDS) proteins are a highly conserved family of transmembrane glycosyltransferases that work in concert with class B penicillin-binding proteins (bPBPs) to build the bacterial peptidoglycan cell wall1,2,3,4,5,6. How these proteins coordinate polymerization of new glycan strands with their crosslinking to the existing peptidoglycan meshwork is unclear. Here, we report the crystal structure of the prototypical SEDS protein RodA from Thermus thermophilus in complex with its cognate bPBP at 3.3 Å resolution. The structure reveals a 1:1 stoichiometric complex with two extensive interaction interfaces between the proteins: one in the membrane plane and the other at the extracytoplasmic surface. When in complex with a bPBP, RodA shows an approximately 10 Å shift of transmembrane helix 7 that exposes a large membrane-accessible cavity. Negative-stain electron microscopy reveals that the complex can adopt a variety of different conformations. These data define the bPBP pedestal domain as the key allosteric activator of RodA both in vitro and in vivo, explaining how a SEDS–bPBP complex can coordinate its dual enzymatic activities of peptidoglycan polymerization and crosslinking to build the cell wall.
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Data availability
Structure factors and refined atomic coordinates for the WT RodA–PBP2 complex and RodA(D255A)–PBP2 variant complex have been deposited in the RCSB PDB under accession codes 6PL5 and 6PL6, respectively. All other data that support the findings of this study are available from the corresponding author upon request. The source data for Figs. 2b and 2d are provided with the paper.
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Acknowledgements
Financial support for the work was provided by National Institutes of Health grant nos. U19AI109764 (A.C.K., D.Z.R., T.G.B., S.W. and D.K.), R01GM106303 (D.S.M.) and 5F31GM128233-02 (S.C.E), the Howard Hughes Medical Institute (T.G.B.) and a Canadian Institutes of Health Research doctoral research award to P.D.A.R. We thank the Advanced Photon Source GM/CA beamline staff for the excellent facilities and technical assistance during X-ray data collection and M. Welsh for the generous gift of purified lipid II substrate.
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M.S. performed the large-scale purification and crystallization of the RodA–PBP2 complexes, the enzymatic assays and negative-stain electron microscopy and data processing. S.C.E. collected the electron microscopy images and provided input on data processing. M.S.A.G. assisted with revisions and performed small-scale protein purification. A.T., D.K. and S.W. provided additional input regarding the enzyme assays. M.S., S.Z. and A.C.K. solved and refined the structure. K.B. and A.G.G., with supervision by D.S.M., performed additional validation of the RodA–PBP2 structure using evolutionary couplings. P.D.A.R. assessed the PBP2 mutant phenotypes with supervision from T.G.B. and D.Z.R. A.C.K. performed the overall project supervision with input from T.G.B. and D.Z.R. M.S. and A.C.K. wrote the manuscript with input from the other authors.
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Extended data
Extended Data Fig. 1 RodA–PBP2 complex interface within the membrane plane.
The transmembrane helix of PBP2 is shown as blue ribbons and transparent molecular surfaces to highlight its interaction with a, RodA transmembrane helix 8 and b, RodA transmembrane helix 9.
Extended Data Fig. 2 Evolutionary covariation analysis of RodA–PBP2 interface.
Graphical representation of 19 evolutionary couplings between RodA-PBP2.
Extended Data Fig. 3 RodA–PBP2 complex interface II.
PBP2 is shown as ribbons colored blue and RodA is shown as molecular surfaces and colored green. The insets show two views of interface II. Side chains of PBP2 residues at the RodA interface are shown as sticks with hydrophobic residues also shown as transparent molecular surface to highlight surface complementarity. Two PBP2 residues that disrupted RodA function when substituted with arginine are labeled.
Extended Data Fig. 4 Shift of TM7 of RodA in complex with PBP2 results in a large membrane-accessible cavity.
Surface view of RodA:PBP2 complex (left) and RodA (right) representing electrostatic potential (top panel) and a cross section (bottom panel). The large surface-exposed cavity is outlined in black dotted lines.
Extended Data Fig. 5 PBP2 mutants co-purify with RodA.
PBP2 variants were co-expressed with FLAG-tagged RodA and purified using anti-FLAG affinity resin. SDS-PAGE gel showing the elution from the anti-FLAG affinity resin demonstrates that PBP2 remains associated with RodA throughout the purification. Results are derived from one experiment.
Extended Data Fig. 6 Structural comparison of extracytoplasmic domains of PBP2 from T. thermophilus and H. pylori.
The catalytic serine in each transpeptidase in shown as a red sphere.
Extended Data Fig. 7 E. coli PBP2 variants co-purify with E. coli RodA.
PBP2 variants were co-expressed with FLAG-tagged RodA and purified using anti-FLAG affinity resin. SDS-PAGE gel showing the elution from the anti-FLAG affinity resin demonstrates that PBP2 remains associated with RodA throughout the purification. Low (L) and high (H) correspond to approximately 2 µg and 4 µg as measured by A280. Results are derived from one experiment.
Extended Data Fig. 8 RodA–PBP2 complex crystal lattice.
The TtRodA:PBP2 complex adopts type 1 lipid cubic phase crystal packing. RodA and PBP2 are shown in green and blue, respectively.
Extended Data Fig. 9 EM analysis of TtRodA–PBP2 complex.
a, Representative EM micrograph of negatively stained RodA:PBP2 complex solubilized in DDM detergent micelles. Scale bar denotes 50 nm. b, Representative two-dimensional class averages from a total of 32,152 particles. Scale bar represents 10 nm. Results are derived from one experiment.
Extended Data Fig. 10 Electron density map.
2Fo−Fc electron density map contoured at 1.0 σ within a 2.3 Å radius of atoms shown for a, the entire T. thermophilus RodA:PBP2 complex b, the transmembrane helix of PBP2 and c, transmembrane helices 5–7 and extracellular loop 4 of RodA.
Supplementary information
Supplementary Information
Supplementary Tables 1 and 2, and Figs. 1–3.
Source data
Source Data Fig. 2
Unprocessed western blots.
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Sjodt, M., Rohs, P.D.A., Gilman, M.S.A. et al. Structural coordination of polymerization and crosslinking by a SEDS–bPBP peptidoglycan synthase complex. Nat Microbiol 5, 813–820 (2020). https://doi.org/10.1038/s41564-020-0687-z
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DOI: https://doi.org/10.1038/s41564-020-0687-z
