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.
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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.
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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.
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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.
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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.
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Raw single-point ELISA and multi-point sAB ELISA data
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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
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DOI: https://doi.org/10.1038/s41586-022-04555-x
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