Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Structural basis of lipopolysaccharide maturation by the O-antigen ligase

Nature volume 604, pages 371–376 (2022)Cite this article

Subjects

Abstract

The outer membrane of Gram-negative bacteria has an external leaflet that is largely composed of lipopolysaccharide, which provides a selective permeation barrier, particularly against antimicrobials1. The final and crucial step in the biosynthesis of lipopolysaccharide is the addition of a species-dependent O-antigen to the lipid A core oligosaccharide, which is catalysed by the O-antigen ligase WaaL2. Here we present structures of WaaL from Cupriavidus metallidurans, both in the apo state and in complex with its lipid carrier undecaprenyl pyrophosphate, determined by single-particle cryo-electron microscopy. The structures reveal that WaaL comprises 12 transmembrane helices and a predominantly α-helical periplasmic region, which we show contains many of the conserved residues that are required for catalysis. We observe a conserved fold within the GT-C family of glycosyltransferases and hypothesize that they have a common mechanism for shuttling the undecaprenyl-based carrier to and from the active site. The structures, combined with genetic, biochemical, bioinformatics and molecular dynamics simulation experiments, offer molecular details on how the ligands come in apposition, and allows us to propose a mechanistic model for catalysis. Together, our work provides a structural basis for lipopolysaccharide maturation in a member of the GT-C superfamily of glycosyltransferases.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: The central role of WaaL in LPS biosynthesis in bacteria and the structure of CmWaaL.
Fig. 2: Key structural features of CmWaaL.
Fig. 3: A putative binding site for lipid A.
Fig. 4: Mechanism of catalysis for WaaL.

Similar content being viewed by others

Data availability

All raw movie frames have been deposited into the Electron Microscopy Public Image Archive (EMPIAR), with accession code EMPIAR-10938. The density maps have been deposited into the Electron Microscopy Data Bank (EMDB), with accession code EMD-26054 for the Und-PP-bound CmWaaL and EMD-26057 for the apo CmWaaL. Both models have been deposited in the Protein Data Bank (PDB), with accession code 7TPG for the Und-PP-bound and 7TPJ for the apo-CmWaaL model. All raw gels are available in the Supplementary Information.

References

  1. Valvano, M. A. Export of O-specific lipopolysaccharide. Front. Biosci. 8, s452–s471 (2003).

    Article  CAS  PubMed  Google Scholar 

  2. Ruan, X., Loyola, D. E., Marolda, C. L., Perez-Donoso, J. M. & Valvano, M. A. The WaaL O-antigen lipopolysaccharide ligase has features in common with metal ion-independent inverting glycosyltransferases. Glycobiology 22, 288–299 (2012).

    Article  CAS  PubMed  Google Scholar 

  3. Whitfield, C. & Trent, M. S. Biosynthesis and export of bacterial lipopolysaccharides. Annu. Rev. Biochem. 83, 99–128 (2014).

    Article  CAS  PubMed  Google Scholar 

  4. Woodward, L. & Naismith, J. H. Bacterial polysaccharide synthesis and export. Curr. Opin. Struct. Biol. 40, 81–88 (2016).

    Article  CAS  PubMed  Google Scholar 

  5. Kaniuk, N. A., Vinogradov, E. & Whitfield, C. Investigation of the structural requirements in the lipopolysaccharide core acceptor for ligation of O antigens in the genus Salmonella: WaaL “ligase” is not the sole determinant of acceptor specificity. J. Biol. Chem. 279, 36470–36480 (2004).

    Article  CAS  PubMed  Google Scholar 

  6. Raetz, C. R., Reynolds, C. M., Trent, M. S. & Bishop, R. E. Lipid A modification systems in gram-negative bacteria. Annu. Rev. Biochem. 76, 295–329 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Hong, Y. & Reeves, P. R. Model for the controlled synthesis of O-antigen repeat units involving the WaaL ligase. mSphere 1, e00074-15 (2016).

    Article  PubMed  Google Scholar 

  8. Lundstedt, E., Kahne, D. & Ruiz, N. Assembly and maintenance of lipids at the bacterial outer membrane. Chem. Rev. 121, 5098–5123 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Whitfield, C., Williams, D. M. & Kelly, S. D. Lipopolysaccharide O-antigens—bacterial glycans made to measure. J. Biol. Chem. 295, 10593–10609 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Liu, B. et al. Structure and genetics of Escherichia coli O antigens. FEMS Microbiol. Rev. 44, 655–683 (2020).

    Article  CAS  PubMed  Google Scholar 

  11. Feldman, M. F. et al. The activity of a putative polyisoprenol-linked sugar translocase (Wzx) involved in Escherichia coli O antigen assembly is independent of the chemical structure of the O repeat. J. Biol. Chem. 274, 35129–35138 (1999).

    Article  CAS  PubMed  Google Scholar 

  12. Bertani, B. & Ruiz, N. Function and biogenesis of lipopolysaccharides. EcoSal Plus 8 (2018).

  13. Schmid, J., Sieber, V. & Rehm, B. Bacterial exopolysaccharides: biosynthesis pathways and engineering strategies. Front. Microbiol. 6, 496 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Ruan, X. & Valvano, M. A. in Glycosyltransferases (ed. Brockhausen, I.) 185–197 (Springer, 2013).

  15. Abeyrathne, P. D., Daniels, C., Poon, K. K., Matewish, M. J. & Lam, J. S. Functional characterization of WaaL, a ligase associated with linking O-antigen polysaccharide to the core of Pseudomonas aeruginosa lipopolysaccharide. J. Bacteriol. 187, 3002–3012 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Pérez, J. M., McGarry, M. A., Marolda, C. L. & Valvano, M. A. Functional analysis of the large periplasmic loop of the Escherichia coli K‐12 WaaL O‐antigen ligase. Mol. Microbiol. 70, 1424–1440 (2008).

    Article  CAS  PubMed  Google Scholar 

  17. Islam, S. T., Taylor, V. L., Qi, M. & Lam, J. S. Membrane topology mapping of the O-antigen flippase (Wzx), polymerase (Wzy), and ligase (WaaL) from Pseudomonas aeruginosa PAO1 reveals novel domain architectures. mBio 1, e00189-00110 (2010).

    Article  CAS  Google Scholar 

  18. Nygaard, R., Kim, J. & Mancia, F. Cryo-electron microscopy analysis of small membrane proteins. Curr. Opin. Struct. Biol. 64, 26–33 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Dominik, P. K. & Kossiakoff, A. A. in Methods in Enzymology Vol. 557 (ed. Shukla, A. K.) 219–245 (Elsevier, 2015).

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

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  21. Newport, T. D., Sansom, M. S. P. & Stansfeld, P. J. The MemProtMD database: a resource for membrane-embedded protein structures and their lipid interactions. Nucleic Acids Res. 47, D390–D397 (2019).

    Article  CAS  PubMed  Google Scholar 

  22. Lazarus, M. B., Nam, Y., Jiang, J., Sliz, P. & Walker, S. Structure of human O-GlcNAc transferase and its complex with a peptide substrate. Nature 469, 564–567 (2011).

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  23. Valvano, M. A. in Recent Trends in Carbohydrate Chemistry (eds Rauter, A. P. et al) 37–49 (Elsevier, 2020).

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

    Article  CAS  Google Scholar 

  25. Meeske, A. J. et al. SEDS proteins are a widespread family of bacterial cell wall polymerases. Nature 537, 634–638 (2016).

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  26. Petrou, V. I. et al. Structures of aminoarabinose transferase ArnT suggest a molecular basis for lipid A glycosylation. Science 351, 608–612 (2016).

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  27. Tavares-Carreón, F., Fathy Mohamed, Y., Andrade, A. & Valvano, M. A. ArnT proteins that catalyze the glycosylation of lipopolysaccharide share common features with bacterial N-oligosaccharyltransferases. Glycobiology 26, 286–300 (2016).

    PubMed  Google Scholar 

  28. Napiórkowska, M. et al. Molecular basis of lipid-linked oligosaccharide recognition and processing by bacterial oligosaccharyltransferase. Nat. Struct. Mol. Biol. 24, 1100–1106 (2017).

    Article  CAS  PubMed  Google Scholar 

  29. Ruan, X., Monjarás Feria, J., Hamad, M. & Valvano, M. A. Escherichia coli and Pseudomonas aeruginosa lipopolysaccharide O‐antigen ligases share similar membrane topology and biochemical properties. Mol. Microbiol. 110, 95–113 (2018).

    Article  CAS  PubMed  Google Scholar 

  30. Voss, N. R. & Gerstein, M. 3V: cavity, channel and cleft volume calculator and extractor. Nucleic Acids Res. 38, W555–W562 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Whitney, J. & Howell, P. Synthase-dependent exopolysaccharide secretion in Gram-negative bacteria. Trends Microbiol. 21, 63–72 (2013).

    Article  CAS  PubMed  Google Scholar 

  32. Whitfield, C. Biosynthesis and assembly of capsular polysaccharides in Escherichia coli. Annu. Rev. Biochem. 75, 39–68 (2006).

    Article  CAS  PubMed  Google Scholar 

  33. Cuthbertson, L., Kos, V. & Whitfield, C. ABC transporters involved in export of cell surface glycoconjugates. Microbiol. Mol. Biol. Rev. 74, 341–362 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Pérez-Burgos, M. et al. Characterization of the exopolysaccharide biosynthesis pathway in Myxococcus xanthus. J. Bacteriol. 202, e00335-20 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Mi, W. et al. Structural basis of MsbA-mediated lipopolysaccharide transport. Nature 549, 233–237 (2017).

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  36. Rizk, S. S. et al. Allosteric control of ligand-binding affinity using engineered conformation-specific effector proteins. Nat. Struct. Mol. Biol. 18, 437 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Miller, K. R. et al. T cell receptor-like recognition of tumor in vivo by synthetic antibody fragment. PLoS ONE 7, e43746 (2012).

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  38. Fellouse, F. A. et al. High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries. J. Mol. Biol. 373, 924–940 (2007).

    Article  CAS  PubMed  Google Scholar 

  39. Punta, M. et al. Structural genomics target selection for the New York consortium on membrane protein structure. J. Struct. Funct. Genomics 10, 255–268 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Mancia, F. & Love, J. High-throughput expression and purification of membrane proteins. J. Struct. Biol. 172, 85–93 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Mancia, F. & Love, J. High throughput platforms for structural genomics of integral membrane proteins. Curr. Opin. Struct. Biol. 21, 517–522 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Bayburt, T. H., Grinkova, Y. V. & Sligar, S. G. Self-assembly of discoidal phospholipid bilayer nanoparticles with membrane scaffold proteins. Nano Lett. 2, 853–856 (2002).

    Article  CAS  ADS  Google Scholar 

  43. Denisov, I. G., Grinkova, Y. V., Lazarides, A. A. & Sligar, S. G. Directed self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size. J. Am. Chem. Soc. 126, 3477–3487 (2004).

    Article  CAS  PubMed  Google Scholar 

  44. Kapust, R. B., Tözsér, J., Copeland, T. D. & Waugh, D. S. The P1′ specificity of tobacco etch virus protease. Biochem. Biophys. Res. Commun. 294, 949–955 (2002).

    Article  CAS  PubMed  Google Scholar 

  45. Dominik, P. K. et al. Conformational chaperones for structural studies of membrane proteins using antibody phage display with nanodiscs. Structure 24, 300–309 (2016).

    Article  CAS  PubMed  Google Scholar 

  46. Kim, J. et al. Structure and drug resistance of the Plasmodium falciparum transporter PfCRT. Nature 576, 315–320 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Suloway, C. et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41–60 (2005).

    Article  CAS  PubMed  Google Scholar 

  48. Slabinski, L. et al. XtalPred: a web server for prediction of protein crystallizability. Bioinformatics 23, 3403–3405 (2007).

    Article  CAS  PubMed  Google Scholar 

  49. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Casañal, A., Lohkamp, B. & Emsley, P. Current developments in Coot for macromolecular model building of electron cryo‐microscopy and crystallographic data. Protein Sci. 29, 1055–1064 (2020).

    Article  CAS  Google Scholar 

  52. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix. refine. Acta Crystallogr. D 68, 352–367 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D 74, 531–544 (2018).

    Article  CAS  Google Scholar 

  54. 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 

  55. Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).

    Article  CAS  PubMed  Google Scholar 

  56. Danilov, L., Druzhinina, T., Kalinchuk, N., Maltsev, S. & Shibaev, V. Polyprenyl phosphates: synthesis and structure-activity relationship for a biosynthetic system of Salmonella anatum O-specific polysaccharide. Chem. Phys. Lipids 51, 191–203 (1989).

    Article  CAS  PubMed  Google Scholar 

  57. Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L. A. RNA-guided editing of bacterial genomes using CRISPR–Cas systems. Nat. Biotechnol. 31, 233–239 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Jiang, Y. et al. Multigene editing in the Escherichia coli genome via the CRISPR–Cas9 system. Appl. Environ. Microbiol. 81, 2506–2514 (2015).

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  59. Selle, K. & Barrangou, R. Harnessing CRISPR–Cas systems for bacterial genome editing. Trends Microbiol. 23, 225–232 (2015).

    Article  CAS  PubMed  Google Scholar 

  60. Jiang, X. et al. Vector promoters used in Klebsiella pneumoniae. Biotechnol. Appl. Biochem. 63, 734–739 (2016).

    Article  CAS  PubMed  Google Scholar 

  61. Zhao, D. et al. Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9. Microb. Cell Fact. 15, 205 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Wang, Y. et al. CRISPR-Cas9 and CRISPR-assisted cytidine deaminase enable precise and efficient genome editing in Klebsiella pneumoniae. Appl. Environ. Microbiol. 84, e01834-01818 (2018).

    Google Scholar 

  63. McConville, T. H. et al. CrrB positively regulates high-level polymyxin resistance and virulence in Klebsiella pneumoniae. Cell Rep. 33, 108313 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Mijnendonckx, K. et al. Characterization of the survival ability of Cupriavidus metallidurans and Ralstonia pickettii from space-related environments. Microb. Ecol. 65, 347–360 (2013).

    Article  CAS  PubMed  Google Scholar 

  65. Schmidt, C., Schwarzenberger, C., Große, C. & Nies, D. H. FurC regulates expression of zupT for the central zinc importer ZupT of Cupriavidus metallidurans. J. Bacteriol. 196, 3461–3471 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Sambrook, J. & Rusell, D. Molecular Cloning: A Laboratory Manual 3rd edn (Cold Spring Harbor Laboratory Press, 2001).

  67. Baba, T. et al. Construction of Escherichia coli K‐12 in‐frame, single‐gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Hitchcock, P. J. & Brown, T. M. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J. Bacteriol. 154, 269–277 (1983).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Remmert, M., Biegert, A., Hauser, A. & Söding, J. HHblits: lightning-fast iterative protein sequence searching by HMM-HMM alignment. Nat. Methods 9, 173–175 (2012).

    Article  CAS  Google Scholar 

  70. Mirdita, M. et al. Uniclust databases of clustered and deeply annotated protein sequences and alignments. Nucleic Acids Res. 45, D170–D176 (2017).

    Article  CAS  PubMed  Google Scholar 

  71. Crooks, G. E., Hon, G., Chandonia, J.-M. & Brenner, S. E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Wu, Q. et al. Protein contact prediction using metagenome sequence data and residual neural networks. Bioinformatics 36, 41–48 (2020).

    Article  CAS  PubMed  Google Scholar 

  73. Robert, X. & Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320–W324 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Šali, A. & Blundell, T. L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993).

    Article  PubMed  Google Scholar 

  75. Abraham, M. J. et al. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1, 19–25 (2015).

    Article  ADS  Google Scholar 

  76. Vogeley, L. et al. Structural basis of lipoprotein signal peptidase II action and inhibition by the antibiotic globomycin. Science 351, 876–880 (2016).

    Article  CAS  ADS  PubMed  Google Scholar 

  77. Wassenaar, T. A., Ingólfsson, H. I., Böckmann, R. A., Tieleman, D. P. & Marrink, S. J. Computational lipidomics with insane: a versatile tool for generating custom membranes for molecular simulations. J. Chem. Theory Comput. 11, 2144–2155 (2015).

    Article  CAS  PubMed  Google Scholar 

  78. Bussi, G., Donadio, D. & Parrinello, M. Canonical sampling through velocity rescaling. J. Chem. Phys. 126, 014101 (2007).

    Article  ADS  CAS  PubMed  Google Scholar 

  79. Parrinello, M. & Rahman, A. Polymorphic transitions in single crystals: a new molecular dynamics method. J. Appl. Phys. 52, 7182–7190 (1981).

    Article  CAS  ADS  Google Scholar 

  80. Stansfeld, P. J. & Sansom, M. S. From coarse grained to atomistic: a serial multiscale approach to membrane protein simulations. J. Chem. Theory Comput. 7, 1157–1166 (2011).

    Article  CAS  PubMed  Google Scholar 

  81. Huang, J. et al. CHARMM36m: an improved force field for folded and intrinsically disordered proteins. Nat. Methods 14, 71–73 (2017).

    Article  CAS  PubMed  Google Scholar 

  82. Miyamoto, S. & Kollman, P. A. Settle: an analytical version of the SHAKE and RATTLE algorithm for rigid water models. J. Comput. Chem. 13, 952–962 (1992).

    Article  CAS  Google Scholar 

  83. Hess, B., Bekker, H., Berendsen, H. J. C. & Fraaije, J. G. E. M. LINCS: a linear constraint solver for molecular simulations. J. Comput. Chem. 18, 1463–1472 (1997).

    Article  CAS  Google Scholar 

  84. Jo, S., Kim, T., Iyer, V. G. & Im, W. CHARMM-GUI: a web-based graphical user interface for CHARMM. J. Comput. Chem. 29, 1859–1865 (2008).

    Article  CAS  PubMed  Google Scholar 

  85. Bonomi, M. et al. Promoting transparency and reproducibility in enhanced molecular simulations. Nat. Methods 16, 670–673 (2019).

    Article  CAS  Google Scholar 

  86. Rubinstein, J. L. & Brubaker, M. A. Alignment of cryo-EM movies of individual particles by optimization of image translations. J. Struct. Biol. 192, 188–195 (2015).

    Article  PubMed  Google Scholar 

  87. Vinés, E. D., Marolda, C. L., Balachandran, A. & Valvano, M. A. Defective O-antigen polymerization in tolA and pal mutants of Escherichia coli in response to extracytoplasmic stress. J. Bacteriol. 187, 3359–3368 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We gratefully acknowledge the assistance of members of the laboratory of F.M., and of the Columbia University cryo-EM facility. We thank G. Davies for his input on the glycosyl transferase mechanism. This work was funded by NIH grants GM132120 (to F.M.), AI150098, AI138576 and AI129940 (to M.S.T.), GM117372 (to A.A.K.), GM116799 (to W. A. Hendrickson), U54 DK104309 (to A.-C.U.), and T32 AI100852 and K08 AI146284 (to T.H.M.). Research in the laboratory of P.J.S. was funded by Wellcome (208361/Z/17/Z), the MRC (MR/S009213/1) and the BBSRC (BB/P01948X/1, BB/R002517/1 and BB/S003339/1). This project made use of time on ARCHER and JADE granted via the UK High-End Computing Consortium for Biomolecular Simulation, HECBioSim (https://www.hecbiosim.ac.uk), supported by the EPSRC (grant no. EP/R029407/1). P.J.S. acknowledges Athena and Sulis at HPC Midlands+, which were funded by the EPSRC on grants EP/P020232/1 and EP/T022108/1, and the University of Warwick Scientific Computing Research Technology Platform for computational access. C.L.B.G. is funded by BBSRC studentship grant BB/M01116X/1 and D.I.R. is funded by a Schaefer Research Scholars Program Awards to Columbia University and MRC grant MR/N002679/1. Some of the work was performed at the Center for Membrane Protein Production and Analysis (COMPPÅ) and at the National Resource for automated Molecular Microscopy at the National Resource for Molecular Microscopy at the Simons Electron Microscopy Center, both located at the New York Structural Biology Center.

Author information

Author notes

  1. These authors contributed equally: Owen N. Vickery, Satchal K. Erramilli, Carmen M. Herrera, Thomas H. McConville

Authors and Affiliations

  1. Department of Physiology and Cellular Biophysics, Columbia University Irving Medical Center, New York, NY, USA

    Khuram U. Ashraf, Rie Nygaard, Sabrina I. Giacometti, Meagan Belcher Dufrisne, Allen P. Zinkle, David I. Roper, Oliver B. Clarke & Filippo Mancia

  2. School of Life Sciences, University of Warwick, Coventry, UK

    Owen N. Vickery, Chris L. B. Graham, David I. Roper & Phillip J. Stansfeld

  3. Department of Chemistry, University of Warwick, Coventry, UK

    Owen N. Vickery & Phillip J. Stansfeld

  4. Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA

    Satchal K. Erramilli, Kamil Nosol & Anthony A. Kossiakoff

  5. Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA, USA

    Carmen M. Herrera & M. Stephen Trent

  6. Department of Medicine, Division of Infectious Diseases, Columbia University Medical Center, New York, NY, USA

    Thomas H. McConville & Anne-Catrin Uhlemann

  7. Department of Microbiology, Biochemistry, and Molecular Genetics, New Jersey Medical School, Rutgers Biomedical Health Sciences, Newark, NJ, USA

    Vasileios I. Petrou

  8. Center for Immunity and Inflammation, New Jersey Medical School, Rutgers Biomedical Health Sciences, Newark, NJ, USA

    Vasileios I. Petrou

  9. New York Consortium on Membrane Protein Structure, New York Structural Biology Center, New York, NY, USA

    Michael Loukeris & Brian Kloss

  10. Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland

    Karolina Skorupinska-Tudek & Ewa Swiezewska

  11. Department of Anesthesiology, Columbia University Irving Medical Center, New York, NY, USA

    Oliver B. Clarke

Authors
  1. Khuram U. Ashraf
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rie Nygaard
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Owen N. Vickery
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Satchal K. Erramilli
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Carmen M. Herrera
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Thomas H. McConville
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Vasileios I. Petrou
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sabrina I. Giacometti
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Meagan Belcher Dufrisne
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Kamil Nosol
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Allen P. Zinkle
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Chris L. B. Graham
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Michael Loukeris
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Brian Kloss
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Karolina Skorupinska-Tudek
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Ewa Swiezewska
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. David I. Roper
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Oliver B. Clarke
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Anne-Catrin Uhlemann
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Anthony A. Kossiakoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. M. Stephen Trent
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Phillip J. Stansfeld
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Filippo Mancia
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.U.A., with help from B.K., M.L., M.B.D. and A.P.Z., performed the genomic expansion screen, protein expression and purification. S.K.E., K.N. and A.A.K. identified and purified the Fabs. K.U.A. produced and analysed the cryo-EM data, and built the model with help from O.B.C. Mutational analyses were performed by K.U.A., V.I.P. and S.I.G. Gene editing for C. metallidurans was performed by T.H.M. and A.-C.U. Assessment of WaaL function was carried out by C.M.H. and M.S.T. All MD simulations were performed by P.J.S. and O.N.V. The ligands for TLC analysis were synthesized by K.S.-T. under the guidance of E.S., and K.U.A. performed the TLC analysis. K.U.A., F.M., P.J.S., O.N.V., C.M.H. and M.S.T. designed experiments and wrote the paper with R.N., C.L.B.G. and D.I.R. Oversight for the entire project was provided by F.M.

Corresponding authors

Correspondence to M. Stephen Trent, Phillip J. Stansfeld or Filippo Mancia.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature thanks Russell Bishop and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Functional validation of CmWaaL, identification of WaaL-specific Fabs and preparation of a nanodisc-reconstituted WaaL–Fab complex for structural analysis.

a, Schematic representation of O-antigen synthesis and transfer to the periplasmic leaflet of the inner membrane by the three different pathways, the arrows represent the direction of the Und-PP linked O-antigen takes in each pathway. Individual lipid-linked O-antigen repeat units are ligated to the lipid carrier Und-PP by glycosyltransferase enzymes. In the Wzy-dependent pathway, the O units are transported into the periplasm by the flippase Wzx. Wzy then catalyses the polymerization of O-antigen repeats, and Wzz controls the preferred modal length of the final O-antigen polymer9. The synthase dependent pathway is the least well characterized pathway31, the O-antigen is assembled at the cytoplasmic face of the inner membrane by a synthase that is also involved in its transportation across the membrane. In the ABC-dependent pathway, the polymerized Und-PP-O-antigen molecule is flipped to the periplasmic face of the inner membrane by an ABC transporter, Wzm-Wzt flippase31,32,33,34. It is important to note that the chemical composition of the C. metallidurans O-antigen is unknown. b, Schematic of WaaL function. On the right, the lipid A core oligosaccharide is synthesized in the cytoplasm and flipped to the periplasm via MsbA35. On the left, the lipid A core oligosaccharide and the O-antigen, irrespective of the pathway of origin, are ligated by WaaL. c, Functional analysis of CmWaaL ligase activity in whole cells. LPS gel showing that O-antigen ligase activity is abolished when Cm waaL is deleted, and activity is restored by plasmid complementation. d, Size-exclusion chromatography elution profiles of purified CmWaaL in detergent (blue), CmWaaL incorporated into a nanodisc (red), and CmWaaL incorporated into a nanodisc with Fab (WaB10) bound (black). e, SDS-PAGE gel of CmWaaL purification. First lane is CmWaaL purified in DDM, second lane is CmWaaL reconstituted into nanodiscs (MSP1E3D1 and POPG), and third lane is CmWaaL reconstituted into nanodiscs (MSP1E3D1 and POPG) with Fab (WaB10) bound. f, Complementarity-determining region (CDR) sequences of unique synthetic antigen binders (sABs) from biopanning against CmWaaL in MSPE3D1 nanodiscs. sABs were selected following multiple rounds of phage display starting from Fab Library E36,37. Enriched YSGW residues are highlighted by coloured boxes (yellow, red, green, and blue, respectively). YSGW residues have been previously shown to play dominant roles in highly specific and high affinity antigen recognition38. g, Single-point ELISA measuring the binding of phage-displayed sABs to CmWaaL in MSP1E3D1 nanodiscs (red), empty nanodiscs (light grey), or buffer (empty wells, dark grey). ELISA signal measured at 450 nm absorbance, see Supplementary Table 1. h, Multi-point sAB ELISA: EC50 estimation for purified sAB binding to CmWaaL incorporated into MSP1E3D1 nanodiscs, showing high affinity binding of WaE8 (green, 6.6 ± 0.045 nM), WaB10 (red, 1.87 ± 0.07 nM), WaC9 (orange, 6.26 ± 0.18 nM), WaG11 (cyan, 3.31 ± 0.06 nM), and WaC10 (magenta, 3.90 ± 0.09 nM), and modest affinity binding of WaF10 (blue, 279.5 ± 0.68 nM) and WaB12 (brown, 154 ± 0.11 nM), see Supplementary Table 1. EC50 values represent the mean of three independent experiments +/- standard error (n = 3).

Extended Data Fig. 2 Cryo-EM analysis of the CmWaaL–Fab complex.

a, Flow chart outlining cryo-EM data processing and refinement performed to obtain a structure of a nanodisc-reconstituted CmWaaL with the Fab WaB10, both for the apo and the Und-PP-bound structures. b, On the left, representative micrograph (2.44 μm defocus). On right, representative two-dimensional class averages from CryoSPARC two-dimensional classification38,86. c, Fourier shell correlation (FSC) curves for the Und-PP-bound CmWaaL–Fab complex. d, Local resolution display of unsharpened reconstructions of Und-PP-bound CmWaaL in complex with the WaB10 Fab, in orthogonal views. e, Euler angle distribution of all Und-PP-bound particles used in the final map reconstruction. Final map shown in green. Each orientation is represented by a cylinder, with each cylinder’s height and colour (from blue to red) proportional to the number of particles for that specific direction.

Extended Data Fig. 3 Fit of cryo-EM density with model.

Cryo-EM densities (mesh) are superimposed on TM and PH helices of the CmWaaL model. The model is rendered as sidechain, coloured in rainbow, as in Fig. 1d. Und-PP (gold) is shown as sticks.

Extended Data Fig. 4 Interaction of CmWaaL and Fab in the complex, and sequence alignment WebLogo.

a, CmWaaL-WaB10 complex structure shown in ribbon with WaaL in grey and WaB10 in pink. Only the variable domain of WaB10 was modelled into the map. b, Interface between CmWaaL (grey) and WaB10 (pink). Residues shown in sticks (I137, D245, S261 for CmWaaL, and N31, Y93, F104 for Fab). c, A WebLogo for orthologues of CmWaaL annotated with TM and soluble helices. The numbering for CmWaaL is shown.

Extended Data Fig. 5 Structural features and analysis of CmWaaL.

a, CmWaaL rendered in surface representation coloured by electrostatic potential on a range of ±5 kBT/e. b, CmWaaL surface coloured by Wimley-White hydrophobicity, on a cyan (very hydrophilic) to gold (very hydrophobic) scale. c, CmWaaL surface coloured by residue conservation on a green (no conservation) to purple (absolute conservation) scale. d, TLC analysis of detergent purified CmWaaL. CmWaaL was purified in detergent and run on the TLC plate after organic-phase extraction of lipids. POPG, Und-P, Und-PP and an unrelated control protein expressed in E. coli were run in separate lanes as standards. MCR-1 was chosen as it uses a lipid donor (phosphatidylethanolamine) to modify the lipid A domain of LPS, but does not use Und-PP. e, Und-PP-binding site. Residues coordinating the first two isoprenyl groups in the Und-PP tail, shown as sticks (blue). Und-PP shown as sticks coloured golden. f, (Top) MD simulations showing the Und-PP binding to CmWaaL from two views, and the flexibility of the Und-PP tail beyond the first two isoprenyl groups. The Und-PP shows increasing mobility away from the pyrophosphate. Here, the root mean squared fluctuation (RMSF) of Und-PP is shown for 3 repeats of 500 ns simulation. The non-hydrogen atoms of Und-PP are coloured by RMSF from blue to red. (Bottom) The RMSF of all atoms of the Und-PP are shown. The portion of the Und-PP resolved in the cryo-EM density map is highlighted in red.

Extended Data Fig. 6 EcWaaL homology model.

a, Co-evolutionary analysis for CmWaaL calculated using MapPred and mapped onto the cryo-EM structure of CmWaaL, using a threshold of 0.272. Predicted contacts between Cα atoms of given residues are shown as dashes for distances less than 10 Å (green), between 10 and 12 Å (cyan), between 12 and 20 Å (yellow), and above 20 Å (red). The CmWaaL structure is shown as a grey ribbon representation. b, Comparison of the CmWaaL structure with the homology model of EcWaaL, both coloured in rainbow from N to C termini. Und-PP is shown bound to both structures and coloured in gold, as well as the key arginine residues that surround Und-PP, show as sticks. c, Sequence alignment and secondary structure of CmWaaL and EcWaaL. Conserved residues are highlighted in black.

Extended Data Fig. 7 Analysis of the ligase activity of CmWaaL and EcWaaL.

a, Functional analysis of EcWaaL ligase activity in whole cells by LPS gel analysis. Ec LPS profile. W3110 ΔwaaL containing either empty vector pWSK29 (Ø), pWSK29::EcWaaL (WT) or pWSK19::EcWaaL-variants87, 101 was evaluated for O-antigen extension. W3110 EcWaaL point mutations that cause loss of ligase activity. b, Table showing key residues in CmWaaL and their corresponding residues in EcWaaL. c, SDS-PAGE gel of all EcWaaL mutants that were purified to verify expression. d, Western blot analysis, using a mouse monoclonal anti-Flag antibody, of Flag purified WT CmWaaL and mutants, grown in C. metallidurans. e, Functional analysis of CmWaaL ligase activity in whole cells by LPS gel analysis C. metallidurans ΔwaaL containing either empty vector pBBR1(Ø), pBBR1:CmWaaL (WT) or pBBR1:CmWaaL-variants was evaluated for O-antigen extension. f, Top view of EcWaaL showing the residues mutated in the two right panels in panel a. g, Top view of the EcWaaL model, highlighting T170 (left panel) and when mutated to Trp (right panel). h, Representative views of lipid A bound to EcWaaL within the interface of cavity 2 (left panel) and an alternative binding site within the Und-PP pocket of cavity 1 (right panel). The lipid A core oligosaccharide is shown as sticks, Und-PP (gold) and H338 are shown as spheres.

Extended Data Fig. 8 Comparison of the Und-PP-bound and the apo-CmWaaL structures.

a, Top Views of the Und-PP bound (grey) and the apo (pink) CmWaaL structures, showing the density (mesh) of the Und-PP (yellow) in the ligand-bound structure in comparison to the apo structure. b, Cryo-EM density maps for the Und-PP-bound (grey) and the apo (pink) CmWaaL. Density maps were prepared in chimeraX55, by deleting any density within a 4 Å radius of the Fab in the final model. c, Side views of the Und-PP-bound (grey) and the apo (pink) CmWaaL shown as ribbon. The Und-PP (yellow) is shown as sticks in the bound structure. d, Top views of the Und-PP-bound (grey) and the apo (pink) CmWaaL showing the key residues that we hypothesize play a role in either binding/shuttling or ligation of the substrates. The density for the selected residues is shown as grey mesh. On the right an overlay of bound and apo states are shown with highlighted residues shown in stick representation.

Extended Data Fig. 9 CmWaaL MD simulations.

a, The RMSF of the backbone of CmWaaL. The RMSF measurements were averaged across 3 repeats of 500 ns simulation. The grey shading refers to the standard deviation across the repeats. Two highly mobile domains are highlighted in yellow (TM9 and PH1) and purple (PH3). These domains have been highlighted on the ribbon structure for reference. The ribbon structures shown are coloured by their respective RMSF from blue to red. b, Representative frames for the closed (red) and open (blue) states are shown, derived from the simulations. The mobility of TM9 is demonstrated by the histograms of the distance between the geometric centres of residues 229–244 and 184–195. The simulations of CmWaaL with Und-PP bound show a stabilization of the closed state, while TM9 separates from the core of CmWaaL in the apo state, opening an access channel to the active site. The initial distance from the cryo-EM structure is highlighted by a black line.

Extended Data Fig. 10 A putative common shuttling mechanism between CmWaaL, RodA, ArnT and PglB.

a, Structural comparison between CmWaaL and TtRodA (PDB ID 6PL5), coloured on a blue to red rainbow from N to C terminus. The two additional helices (TM1 and TM2) of CmWaaL are in grey. b, A comparison of the putative access pathway for polyprenyl-linked-phosphate containing ligands for CmWaaL, TtRodA, CmArnT (PDB ID 5F15) and ClPglB (PDB ID 5OGL). The equivalent TM helix is shown in red and periplasmic helix in blue. Conserved residues, that may be involved in the shuttling and/or coordination of polyprenyl-phosphate-containing ligands in CmWaaL, TtRodA, CmArnT and ClPglB are shown in sticks and highlighted in blue.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
Full size table

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-4

Reporting Summary

Peer Review File

Supplementary Table 1

Raw single-point ELISA and multi-point sAB ELISA data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ashraf, K.U., Nygaard, R., Vickery, O.N. et al. Structural basis of lipopolysaccharide maturation by the O-antigen ligase. Nature 604, 371–376 (2022). https://doi.org/10.1038/s41586-022-04555-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-022-04555-x

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing