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Cryo-EM structure of the bacterial divisome core complex and antibiotic target FtsWIQBL

Nature Microbiology volume 8, pages 1149–1159 (2023)Cite this article

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Abstract

In most bacteria, cell division relies on the synthesis of new cell wall material by the multiprotein divisome complex. Thus, at the core of the divisome are the transglycosylase FtsW, which synthesises peptidoglycan strands from its substrate Lipid II, and the transpeptidase FtsI that cross-links these strands to form a mesh, shaping and protecting the bacterial cell. The FtsQ–FtsB–FtsL trimeric complex interacts with the FtsWI complex and is involved in regulating its enzymatic activities; however, the structure of this pentameric complex is unknown. Here, we present the cryogenic electron microscopy structure of the FtsWIQBL complex from Pseudomonas aeruginosa at 3.7 Å resolution. Our work reveals intricate structural details, including an extended coiled coil formed by FtsL and FtsB and the periplasmic interaction site between FtsL and FtsI. Our structure explains the consequences of previously reported mutations and we postulate a possible activation mechanism involving a large conformational change in the periplasmic domain. As FtsWIQBL is central to the divisome, our structure is foundational for the design of future experiments elucidating the precise mechanism of bacterial cell division, an important antibiotic target.

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Fig. 1: Biochemical and structural characterization of the core divisome complex FtsWIQBL from P. aeruginosa.
Fig. 2: Interactions between FtsI, FtsQ, FtsB and FtsL.
Fig. 3: Interactions that affect divisome regulation.

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Data availability

The final cryo-EM map has been deposited in the Electron Microscopy Data Bank under accession no. EMD-16042. The final model has been deposited with the PDB under accession no. 8BH1. The PDB entries 6H9N, 3PBN, 5Z2W, 6BAS, 6PL5, 3OCN were used for structural superposition, analyses and pixel size calibration. Source data are provided with this paper.

References

  1. Daley, D. O., Skoglund, U. & Söderström, B. FtsZ does not initiate membrane constriction at the onset of division. Sci. Rep. 6, 33138 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Lutkenhaus, J., Pichoff, S. & Du, S. Bacterial cytokinesis: from Z ring to divisome. Cytoskeleton 69, 778–790 (2012).

    Article  CAS  PubMed  Google Scholar 

  3. Taguchi, A. et al. FtsW is a peptidoglycan polymerase that is functional only in complex with its cognate penicillin-binding protein. Nat. Microbiol. 4, 587–594 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Kumar, S., Mollo, A., Kahne, D. & Ruiz, N. The bacterial cell wall: from Lipid II flipping to polymerization. Chem. Rev. 122, 8884–8910 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Egan, A. J., van’t Veer, I., Breukink, E. & Vollmer, W. Activities and regulation of peptidoglycan synthases.Philos. Trans. R Soc. Lond. B Biol. Sci. 370, 20150031 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Egan, A. J. F., Errington, J. & Vollmer, W. Regulation of peptidoglycan synthesis and remodelling. Nat. Rev. Microbiol. 18, 446–460 (2020).

    Article  CAS  PubMed  Google Scholar 

  7. Marmont, L. S. & Bernhardt, T. G. A conserved subcomplex within the bacterial cytokinetic ring activates cell wall synthesis by the FtsW-FtsI synthase. Proc. Natl Acad. Sci. USA 117, 23879–23885 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Liu, Y. & B, E. The membrane steps of bacterial cell wall synthesis as antibiotic targets. Antibiotics 5, 28 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Lomize, M. A., Pogozheva, I. D., Joo, H., Mosberg, H. I. & Lomize, A. L. OPM database and PPM web server: resources for positioning of proteins in membranes. Nucleic Acids Res. 40, D370–D376 (2012).

    Article  CAS  PubMed  Google Scholar 

  10. van den Ent, F. et al. Structural and mutational analysis of the cell division protein FtsQ. Mol. Microbiol. 68, 110–123 (2008).

    Article  CAS  PubMed  Google Scholar 

  11. Kureisaite-Ciziene, D. et al. Structural analysis of the interaction between the bacterial cell division proteins FtsQ and FtsB.mBio 9, e01346-18 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Choi, Y. et al. Structural insights into the FtsQ/FtsB/FtsL complex, a key component of the divisome. Sci. Rep. 8, 18061 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Khadria, A. S. & Senes, A. The transmembrane domains of the bacterial cell division proteins FtsB and FtsL form a stable high-order oligomer. Biochemistry 52, 7542–7550 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Condon, S. G. F. et al. The FtsLB subcomplex of the bacterial divisome is a tetramer with an uninterrupted FtsL helix linking the transmembrane and periplasmic regions. J. Biol. Chem. 293, 1623–1641 (2018).

    Article  CAS  PubMed  Google Scholar 

  15. LaPointe, L. M. et al. Structural organization of FtsB, a transmembrane protein of the bacterial divisome. Biochemistry 52, 2574–2585 (2013).

    Article  CAS  PubMed  Google Scholar 

  16. Robichon, C., Karimova, G., Beckwith, J. & Ladant, D. Role of leucine zipper motifs in association of the Escherichia coli cell division proteins FtsL and FtsB. J. Bacteriol. 193, 4988–4992 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Buddelmeijer, N. & Beckwith, J. A complex of the Escherichia coli cell division proteins FtsL, FtsB and FtsQ forms independently of its localization to the septal region. Mol. Microbiol. 52, 1315–1327 (2004).

    Article  CAS  PubMed  Google Scholar 

  18. Sjodt, M. et al. Structural coordination of polymerization and crosslinking by a SEDS–bPBP peptidoglycan synthase complex. Nat. Microbiol 5, 813–820 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Park, K.-T., Du, S. & Lutkenhaus, J. Essential role for FtsL in activation of septal peptidoglycan synthesis. mBio 11, e03012-20 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Han, S. et al. Structural basis for effectiveness of siderophore-conjugated monocarbams against clinically relevant strains of Pseudomonas aeruginosa. Proc. Natl Acad. Sci. USA 107, 22002–22007 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Paulussen, F. M. et al. Covalent proteomimetic inhibitor of the bacterial FtsQB divisome complex. J. Am. Chem. Soc. 144, 15303–15313 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Egan, A. J., Cleverley, R. M., Peters, K., Lewis, R. J. & Vollmer, W. Regulation of bacterial cell wall growth. FEBS J. 284, 851–867 (2017).

    Article  CAS  PubMed  Google Scholar 

  23. Sjodt, M. et al. Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis. Nature 556, 118–121 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Shlosman, I. et al. Allosteric activation of cell wall synthesis during bacterial growth. Preprint at bioRxiv https://doi.org/10.1101/2022.11.07.515454 (2022).

  26. Gerding, M. A. et al. Self-enhanced accumulation of FtsN at division sites and roles for other proteins with a SPOR domain (DamX, DedD, and RlpA) in Escherichia coli cell constriction. J. Bacteriol. 191, 7383–7401 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Weiss, D. S. Last but not least: new insights into how FtsN triggers constriction during Escherichia coli cell division. Mol. Microbiol. 95, 903–909 (2015).

    Article  CAS  PubMed  Google Scholar 

  28. Wissel, M. C. & Weiss, D. S. Genetic analysis of the cell division protein FtsI (PBP3): amino acid substitutions that impair septal localization of FtsI and recruitment of FtsN. J. Bacteriol. 186, 490–502 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Chen, J. C. & Beckwith, J. FtsQ, FtsL and FtsI require FtsK, but not FtsN, for co-localization with FtsZ during Escherichia coli cell division. Mol. Microbiol. 42, 395–413 (2001).

    Article  PubMed  Google Scholar 

  30. Addinall, S. G., Cao, C. & Lutkenhaus, J. FtsN, a late recruit to the septum in Escherichia coli. Mol. Microbiol. 25, 303–309 (1997).

    Article  CAS  PubMed  Google Scholar 

  31. Liu, B., Persons, L., Lee, L. & de Boer, P. A. J. Roles for both FtsA and the FtsBLQ subcomplex in FtsN-stimulated cell constriction in Escherichia coli. Mol. Microbiol. 95, 945–970 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Tsang, M.-J. & Bernhardt, T. G. A role for the FtsQLB complex in cytokinetic ring activation revealed by an ftsL allele that accelerates division. Mol. Microbiol. 95, 925–944 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Ghigo, J. M. & Beckwith, J. Cell division in Escherichia coli: role of FtsL domains in septal localization, function, and oligomerization. J. Bacteriol. 182, 116–129 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Li, Y. et al. Genetic analysis of the septal peptidoglycan synthase FtsWI complex supports a conserved activation mechanism for SEDS-bPBP complexes. PLoS Genet. 17, e1009366 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Goehring, N. W., Gueiros-Filho, F. & Beckwith, J. Premature targeting of a cell division protein to midcell allows dissection of divisome assembly in Escherichia coli. Genes Dev. 19, 127–137 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Hopf, T. A. et al. The EVcouplings Python framework for coevolutionary sequence analysis. Bioinformatics 35, 1582–1584 (2019).

    Article  CAS  PubMed  Google Scholar 

  37. Weissmann, F. et al. biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes. Proc. Natl Acad. Sci. USA 113, E2564–E2569 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. van den Berg van Saparoea, H. B. et al. Fine-mapping the contact sites of the Escherichia coli cell division proteins FtsB and FtsL on the FtsQ protein. J. Biol. Chem. 288, 24340–24350 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Bieniossek, C., Richmond, T. J. & Berger, I. MultiBac: multigene baculovirus-based eukaryotic protein complex production. Curr. Protoc. Protein Sci. Chapter 5, Unit 5.20 (2008).

    PubMed  Google Scholar 

  40. Laverty, D. et al. Cryo-EM structure of the human α1β3γ2 GABAA receptor in a lipid bilayer. Nature 565, 516–520 (2019).

    Article  CAS  PubMed  Google Scholar 

  41. Ritchie, T. K. et al. Chapter 11—reconstitution of membrane proteins in phospholipid bilayer nanodiscs. Methods Enzymol. 464, 211–231 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Qiao, Y. et al. Detection of lipid-linked peptidoglycan precursors by exploiting an unexpected transpeptidase reaction. J. Am. Chem. Soc. 136, 14678–14681 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Scheres, S. H. W. A Bayesian view on cryo-EM structure determination. J. Mol. Biol. 415, 406–418 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  47. Bepler, T. et al. Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs. Nat. Methods 16, 1153–1160 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  49. Mirdita, M. et al. ColabFold: making protein folding accessible to all. Nat. Methods 19, 679–682 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Turk, D. MAIN software for density averaging, model building, structure refinement and validation. Acta Crystallogr. D Biol. Crystallogr. 69, 1342–1357 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D Struct. Biol. 75, 861–877 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).

    Article  CAS  PubMed  Google Scholar 

  54. Welsh, M. A. et al. Identification of a functionally unique family of penicillin-binding proteins. J. Am. Chem. Soc. 139, 17727–17730 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Schneider, T. et al. In vitro assembly of a complete, pentaglycine interpeptide bridge containing cell wall precursor (lipid II-Gly5) of Staphylococcus aureus. Mol. Microbiol. 53, 675–685 (2004).

    Article  CAS  PubMed  Google Scholar 

  56. Barrett, D. et al. Analysis of glycan polymers produced by peptidoglycan glycosyltransferases. J. Biol. Chem. 282, 31964–31971 (2007).

    Article  CAS  PubMed  Google Scholar 

  57. Salje, J. & Löwe, J. Bacterial actin: architecture of the ParMRC plasmid DNA partitioning complex. EMBO J. 27, 2230–2238 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  PubMed  Google Scholar 

  59. Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).

    Article  CAS  PubMed  Google Scholar 

  60. Agulleiro, J. I. & Fernandez, J. J. Fast tomographic reconstruction on multicore computers. Bioinformatics 27, 582–583 (2011).

    Article  CAS  PubMed  Google Scholar 

  61. Ashkenazy, H. et al. ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Res. 44, W344–W350 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank all members of the MRC LMB electron microscopy facility for excellent electron microscopy support and T. Darling and J. Grimmett for computing support. We thank D. Ritson and M. Su for their help and advice during the Lipid II preparation and H. Kramer (1979–2022) for the liquid chromatography–mass spectrometry and matrix-assisted laser desorption/ionization coupled to time-of-flight mass spectrometry of the Lipid II samples (all MRC LMB). N-terminal His-tagged SaPBP4 (amino acids 21–383) was a gift of the Walker laboratory (Harvard). We thank S. Nayak (MRC LMB VisLabs) for her help with designing the figures. We thank T. Bharat and S. Tetter for their feedback on the manuscript. This work was supported by the Volkswagen Stiftung ‘Life?’ programme (to J.L.) and by the MRC as part of United Kingdom Research and Innovation. MRC file reference no. U105184326 (to J.L.). For the purpose of open access, the MRC Laboratory of Molecular Biology has applied a CC BY public copyright licence to any author-accepted manuscript version arising.

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  1. These authors contributed equally: Lisa Käshammer, Fusinita van den Ent.

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  1. Medical Research Council Laboratory of Molecular Biology, Cambridge, UK

    Lisa Käshammer, Fusinita van den Ent, Magnus Jeffery, Nicolas L. Jean, Victoria L. Hale & Jan Löwe

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Contributions

L.K., F.v.d.E. and M.J. purified the PaFtsQBLWI complex. L.K. collected and processed the cryo-EM data of the PaFtsQBLWI complex. F.v.d.E. and M.J. purified the EcFtsQBLWI complex. M.J. collected and processed the cryo-EM data of the EcFtsQBLWI complex. F.v.d.E. purified Lipid II and performed the FtsWIQBL activity assays. N.L.J. cloned and purified the FtsQ–FtsK complex. V.L.H. prepared, measured and processed the tomogram. J.L. provided the concept and L.K., M.J. and J.L. prepared the manuscript. All authors contributed to the Methods section and to editing the manuscript.

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Correspondence to Jan Löwe.

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Extended data

Extended Data Fig. 1 Protein purification and grid preparation of FtsWIQBL from E. coli (Ec) and P. aeruginosa (Pa).

a) SDS-PAGE gel of the co-expressed and purified EcFtsWIQBL divisome core complex after size-exclusion chromatography in GDN. FtsW and FtsI are joined by a short linker in this construct. b) Size-exclusion chromatogram of EcFtsWIQBL reconstituted in nanodiscs. The measured absorbances at 260 nm and 280 nm are shown in orange and blue, respectively. The peak fraction of the size exclusion run, indicated with an asterisk, was used for grid preparation. c) Representative micrograph of EcFtsWIQBL in nanodiscs. d) Representative 2D Classes of EcFtsWIQBL in nanodiscs. e) Size-exclusion chromatogram of PaFtsWIQBL. The measured absorbances at 260 nm and 280 nm are shown in orange and blue, respectively. The peak fraction of the size exclusion run, indicated with an asterisk, was used for grid preparation. f) Representative micrograph of PaFtsWIQBL used for the final reconstruction.

Source data

Extended Data Fig. 2 Details of the PaFtsWIQBL cryo-EM processing.

a) Cryo-EM image processing scheme for PaFtsWIQBL. b) Fourier Shell Correlation (FSC) curves for the PaFtsWIQBL cryo-EM maps and structures. c) The local resolution of the final structure of PaFtsWIQBL was calculated with RELION’s own implementation and depicted in ChimeraX53. d) The angular distribution of the particles making up the final structure of PaFtsWIQBL.

Extended Data Fig. 3 Details of the cryo-EM density around helices of PaFtsWIQBL.

All TM helices visible in the structure are shown, as well as the FtsLα1 and FtsB α2 coils, two helices from FtsQβ and two helices from the FtsITP domain.

Extended Data Fig. 4 Architecture of the PaFtsWIQBL complex.

a) Upper left panel: schematic of the transmembrane helices of FtsW, FtsI, FtsL and FtsB. Two extracellular loops of FtsW that could not be build due to missing density and the N- and C-terminal tails of FtsW are indicated by doted lines. Lower left panel: top view of the transmembrane domain, with FtsW transmembrane helices consecutively numbered based on the sequence (identical to numbering of helices in a previous RodA structure23). FtsW’s putative active site residue D275 is indicated. Right panel: Labelling of the different domains in FtsQ, FtsL, FtsB and FtsI that was used throughout the paper. b) Cryo-EM density showing PaFtsWIQBL within the Lauryl Maltose Neopentyl Glycol (LMNG) detergent micelle, which was subtracted during the later processing stages. c) Prediction of the position and orientation of the divisome core complex transmembrane segments in the lipid bilayer using the Orientations of Proteins in Membranes webserver9. The membrane plane is indicated with two grey discs and the active sites of FtsW and FtsI are labelled. d) A low-resolution structure obtained after fewer 3D classifications shows additional density for FtsQPOTRA at low contour levels and indicates that the transmembrane segment of FtsQ is most likely not part of the micelle that contains the other TM segments. Alignment of a previous FtsB:FtsQ crystal structure (PDB: 6H9N) on FtsQβ shows that FtsQβ and FtsQPOTRA adopt different conformations relative to each other.

Extended Data Fig. 5 Detailed analysis of the interactions between FtsB and FtsL, FtsW and FtsL and FtsI and FtsL.

a) Interaction sites between FtsB and FtsL, as also indicated in Fig. 2a. Residues of FtsL that also interact with FtsI are highlighted in blue. b) Analysis of the interaction sites between FtsL and FtsW in their transmembrane region. The coiled coil conformation of FtsL means that the interaction surface is not as extended as it would be if it were straighter and not in a coiled coil. c) Electrostatic analysis of the interactions between FtsI and FtsL shows that the interaction site in the coiled coil area is mainly hydrophobic/neutral.

Extended Data Fig. 6 Comparison of PaFtsWIQBL cryo-EM structure with previous crystal structures of FtsI, FtsQ and RodA.

a) Superposition of FtsI determined by X-ray crystallography (PDB: 3PBN, grey) with the FtsI part of PaFtsWIQBL cryo-EM structure. The TP active site residue S294 is indicated (RMSD of 0.708 Å across 372 pruned atom pairs). b) Superposition of FtsQB determined by X-ray crystallography (PDB: 6H9N in dark grey, PDB: 5Z2W in light grey) with the same area in the cryo-EM structure determined here (For alignment of FtsQ: RMSD (FtsQ-6H9N) of 1.186 Å across 86 pruned atom pairs, RMSD (FtsQ-5Z2W) of 1.118 Å across 95 pruned atom pairs). c) Superposition of RodA determined by X-ray crystallography (PDB: 6BAS in dark grey (left and right), PDB: 6PL5 in light gray (right)) and FtsW in the cryo-EM structure. The position of FtsI is indicated as a transparent blue outline. Apart from transmembrane helix 7, the structures align very well (RMSD (FtsW-6PL5) of 1.188 Å across 202 pruned atom pairs; RMSD (FtsW-6BAS) of 1.126 Å across 206 pruned atom pairs). d) Electrostatic surface representation of PaFtsW viewed from the periplasmic side. A deep cleft is visible that contains the putative active site residue D275. The same representation showing sequence conservation of FtsW mapped onto the surface representation shows that this cleft is highly conserved. Additionally, interaction sites with FtsI and FtsL are indicated; these also show above average levels of sequence conservation.

Extended Data Fig. 7 Comparison of the PaFtsWIQBL cryo-EM structure with a RodA-PBP2 crystal structure and AlphaFold 2 structure prediction.

Comparison of the cryo-EM structure PaFtsWIQBL (a), the Thermus thermophilus RodA-PBP2 crystal structure (PDB: 6PL5, b) and the AlphaFold 2 prediction of PaFtsWIQBL (c). All three structures were aligned on FtsW/RodA. The FtsQPOTRA and FtsQTM of the AlphaFold2 model were removed for clarity. The FtsI/PBP2 periplasmic domains show a large 130° lateral rotation between the P. aeruginosa FtsWI and T. thermophilus RodA-PBP2 models (a-b). The rotation was measured around an axis perpendicular to the membrane plane and intersecting the FtsW active site. The distance between both active sites in FtsI (S294) and PBP2 (S308) is 125 Å. A 30° vertical rotation of the periplasmic FtsI domains is visible between the cryo-EM and AlphaFold2 models of PaFtsWIQBL (a-c). The angle was measured between the FtsW active site (D275) and the FtsI active sites (S294). The distance between the FtsI active sites in the cryo-EM and AlphaFold2 models is 46 Å.

Extended Data Fig. 8 Interactions of FtsQ with FtsK.

a) AlphaFold2 model of E. coli FtsWIQBL + FtsK N-terminal domain (residues 1-222; FtsK1–222, grey), showing a predicted interaction between FtsQPOTRA and a periplasmic loop from FtsK (FtsKW51-H57). b) Co-evolutionary coupling analysis calculated with EVcouplings36 finds six out of the ten residue pairs located between FtsQPOTRA and FtsKW51-H57 in the AF2 model in a): FtsQV109 – FtsKW56 (blue), FtsQQ96 – FtsKT54 (dark blue), FtsQS110 – FtsKQ53 (gray), FtsQV111 – FtsKT54 (dark blue), FtsQR112 – FtsKQ53 (grey), and FtsQK113-FtsKS52 (blue). c) Sequence conservation analysis (calculated using ConSurf webserver61) of the same area shows that the β-strands of FtsQ and FtsKW51-H57 that are predicted to interact are highly conserved. Amino acid residues that abolish FtsQ localisation (which is dependent on FtsK septum localisation in cells) when mutated are shown as sticks and are labelled10. d) Size-exclusion trace and SDS-PAGE gel of the co-expression and purification of E. coli FtsQ and FtsK1–222 shows clear co-migration of the proteins.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1–4.

Reporting Summary

Supplementary Video 1

Architecture of the PaFtsWIQBL complex. The position of PaFTSWIQBL in the membrane was calculated with the Orientations of Proteins in Membranes Web server and is indicated as grey planes.

Supplementary Video 2

Morph between the PaFtsWIQBL cryo-EM structure and the PaFtsWIQBL AlphaFold 2 prediction.

Supplementary Data 1

Plasmid maps

Source data

Source Data Fig. 1

Unprocessed SDS–PAGE and activity assay for Fig. 1b,c.

Source Data Extended Data Fig. 1

Unprocessed SDS–PAGE for Extended Data Fig. 1a.

Source Data Extended Data Fig. 8

Unprocessed SDS–PAGE for Extended Data Fig. 8d.

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Käshammer, L., van den Ent, F., Jeffery, M. et al. Cryo-EM structure of the bacterial divisome core complex and antibiotic target FtsWIQBL. Nat Microbiol 8, 1149–1159 (2023). https://doi.org/10.1038/s41564-023-01368-0

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