Outer membrane utilisomes mediate glycan uptake in gut Bacteroidetes

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Bacteroidetes are abundant members of the human microbiota, utilizing a myriad of diet- and host-derived glycans in the distal gut1. Glycan uptake across the bacterial outer membrane of these bacteria is mediated by SusCD protein complexes, comprising a membrane-embedded barrel and a lipoprotein lid, which is thought to open and close to facilitate substrate binding and transport. However, surface-exposed glycan-binding proteins and glycoside hydrolases also play critical roles in the capture, processing and transport of large glycan chains. The interactions between these components in the outer membrane are poorly understood, despite being crucial for nutrient acquisition by our colonic microbiota. Here we show that for both the levan and dextran utilization systems of Bacteroides thetaiotaomicron, the additional outer membrane components assemble on the core SusCD transporter, forming stable glycan-utilizing machines that we term utilisomes. Single-particle cryogenic electron microscopy structures in the absence and presence of substrate reveal concerted conformational changes that demonstrate the mechanism of substrate capture, and rationalize the role of each component in the utilisome.

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The data supporting the findings of this study are available from the corresponding authors upon reasonable request. Cryo-EM reconstructions and corresponding coordinates have been deposited in the Electron Microscopy Data Bank and the PDB, respectively: substrate-free levan utilisome (EMD-15288 and PDB 8A9Y), levan utilisome with FOS DP 8–12 (EMD-15289 and PDB 8AA0), SusC2D2 core from the levan utilisome with FOS DP 8–12 (EMD-15290 and PDB 8AA1), inactive levan utilisome with FOS DP 15–25 (EMD-15291 and PDB 8AA2), SusC2D2 core from inactive levan utilisome with FOS DP 15–25 (EMD-1592 and PDB 8AA3), dextran utilisome consensus refinement (EMD-15293 and PDB 8AA4). Coordinates and structure factors from X-ray crystallography experiments for GHlev have been deposited in the PDB under the accession codes 7ZNR and 7ZNS. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD034863. Raw data from this study are available at the University of Leeds Data Repository: https://doi.org/10.5518/1329. Source data are provided with this paper.

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J.B.R.W. was supported by a Wellcome Trust 4-year PhD studentship (215064/Z/18/Z). B.v.d.B. is financially supported by a Wellcome Trust Investigator award (214222/Z/18/Z), supporting A.S. and Y.Z. M.F. is supported by a Newcastle University studentship. We acknowledge the Diamond Light Source (Didcot, UK) for beam time (proposals mx306, mx1221, mx13587 and mx18598), and thank the staff of beamlines I02, I03, I04 and I24 for support. All cryo-EM was carried out at the Astbury Biostructure Laboratory, which was financially supported by the University of Leeds and the Wellcome Trust (108466/Z/15/Z and 221524/Z/20/Z). We thank R. Thompson, E. Hesketh and D. Maskell for electron microscopy support. Protein ID mass spectrometry was carried out at the University of Leeds Mass Spectrometry Facility. We thank J. Ault and R. George for carrying out this analysis. For the purpose of open access, the authors have applied a CC BY public copyright licence to any author accepted manuscript version arising from this submission.

These authors contributed equally: Joshua B. R. White, Augustinas Silale

Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK

Joshua B. R. White & Neil A. Ranson

Biosciences Institute, The Medical School, Newcastle University, Newcastle upon Tyne, UK

Augustinas Silale, Matthew Feasey, Tiaan Heunis, Yiling Zhu, Hong Zheng, Akshada Gajbhiye, Susan Firbank, Arnaud Baslé, Matthias Trost, David N. Bolam & Bert van den Berg

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J.B.R.W. carried out cryo-EM and determined cryo-EM structures, supervised by N.A.R. A.S. purified proteins, determined X-ray crystal structures and carried out ITC, supervised by B.v.d.B. M.F. purified proteins. Y.Z. prepared OM samples for proteomics. T.H. and A.G. carried out proteomics, supervised by M.T. H.Z. purified Bt1760, supervised by D.N.B. S.F. collected X-ray crystallography data for Bt1760. A.B. solved Bt1760 crystal structures and managed the Newcastle Structural Biology Laboratory. B.v.d.B. generated B. theta strains, purified proteins and crystallized Bt1761. J.B.R.W., A.S., D.N.B., B.v.d.B. and N.A.R. wrote the manuscript.

Correspondence to Bert van den Berg or Neil A. Ranson.

The authors declare no competing interests.

Nature thanks Mirjam Czjzek, Stephen Withers and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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

(a) Organisation of the levan PUL showing relative gene positions within the PUL, with functions indicated. The four OM-associated PUL components (SusClev, SusDlev, GHlev and SGBPlev) are highlighted by the grey box. An X-ray structure of GHlev (Bt1760; GH32 endo-levanase) is shown (blue; PDB-ID: 7ZNR). The AlphaFold2-predicted model for SGBPlev (Bt1761) is shown (pink) oriented such that the N-terminus is at the bottom and the proposed (C-terminal) levan binding domain is at the top. Note that the N-termini of GHlev and SGBPlev will be lipidated and associated with the outer leaflet of the OM. The cryo-EM structure of the dimeric SusCDlev complex in its open-open state is shown (SusClev is green, SusDlev is grey). (b) Organisation of the dextran PUL showing gene positions within the locus with functions labelled. OM-associated PUL components are boxed in grey. AlphaFold2-predicted models for GHdex (Bt3087; GH66 endo-dextranase), the putative SGBPdex (Bt3088) and the SusCDdex complex, are shown coloured as for the levan PUL in (a). (c) SDS-PAGE of the previously-studied sample of LDAO-purified SusCDlev10 before (asterisk) and after boiling. The boiled sample shows two weak bands in addition to those for SusClev and SusDlev, which were subsequently identified as GHlev and SGBPlev by mass spectrometry. (d) A class average obtained during 3D classification of the levan SusC2D2 core complex. The SusC and SusD components (green and grey respectively) are docked into the density. A large region of density remains unassigned (orange). (e) Isolated view of the previously unassigned density with the crystal structure of GHlev (blue cartoon) fitted into the EM density (blue) as a rigid body. The remaining density was therefore attributed to SGBPlev and is coloured magenta.

Source data

(a) Output of the first round of 3D classification for apo utilisome data. Yellow, purple and pink classes represent the octameric complex i.e. the complete octameric utilisome. The green class shows the additional lipoproteins associated with just one SusC unit whilst the blue class shows that a small proportion of SusC2D2 core complex was present. (b) Output of 3D classification for the levan utilisome with an active levanase in the presence of FOS DP8-12. Classes (viewed in the plane of the membrane) containing particles of the complete octameric complex were observed (blue and green) as well as hexameric complexes containing a single copy of the SGBPlev and GHlev (pink and yellow). A class containing SusCDlev in isolation is also present (purple). (c) Outputs of 3D classification for long FOS (DP15-25) showing that SGBPlev can adopt a ‘docked’ conformation proximal to both the SusD and levanase. (d) A consensus refinement of all classes containing at least one docked SGBP (yellow, pink, cyan and green in panel (c)). A mask was created around the region of interest (transparent yellow). (e) Outputs of focused classification on the masked region without alignment. A class displaying high resolution for the region of interest is marked with a red asterisk. Independent half maps were reconstructed using unmasked particles belonging to this class. (f) Sharpened reconstruction generated with the aforementioned half maps showing improved density for SGBPlev.

(a) 3D classification of apo levan utilisome viewed from outside the cell. SusClev (green), SusDlev (grey), SGBPlev (magenta), and GHlev (blue). Classes are separated on their SusDlev lid positions. Wide-wide (WW), normal-wide (NW), and normal-normal (NN) open states (from left to right) (b) Overlay of the wide (SusD grey) and normal (SusD orange) open states of the complex. (c) Overlay of atomic models for the normal versus wide open state generated by a rigid-body fit of SusDlev into the cryoEM density. A monomer is shown for clarity and an asterisk marks the same SusDlev helix in both models. (d) A view of the utilisomes shown at high threshold in the plane of the membrane (left). Different conformations of the SGBPlev observed in 3D classification are overlaid to demonstrate the flexibility of this subunit (boxed region). The same view rotated 90° is shown (right). Disordered micelle density is shown as translucent grey. (e) Variability of the SGBPlev position in the substrate-bound utilisomes with short FOS (~DP8-12) and an active GHlev, and long FOS (DP15-25) with an inactive GHlev. A novel state is uniquely observed in the long FOS structure with one SGBPlev (orange) reaching across and contacting the SGBPlev associated with the other SusC subunit that is present in a docked state. This conformation is consistent with both SGBPlev subunits in the utilisome interacting with the same chain of substrate.

a, Arrangement of GHlev and SGBPlev on SusC in the levan utilisome. GHlev makes contacts with extracellular loop 1 (gold) and extracellular loop 9 (red), while SGBPlev only makes contacts with extracellular loop 1. b, Arrangement of GHdex and SGBPdex on SusC in the dextran utilisome. Here, extracellular loop 1 of SusCdex is the primary site of interaction for GHdex, while extracellular loop 9 comprises the interface with SGBPdex. For clarity, one half of the utilisome is shown in each case, and SusD components are omitted. Note that the dextran utilisome model is a composite of cryo-EM structures (SusCdex) and predicted models from AlphaFold2 (GHdex and SGBPdex).

Side (a) and top (b) view of the heptameric dextran utilisome map. The identical side (c) and top (d) views of a composite atomic model for dextran utilisome is shown. CryoEM data permitted refinement of SusCdex. AlphaFold2 structure predictions for SusDdex and GHdex were docked into the cryoEM map for the heptameric complex. An AlphaFold2 structure prediction for part of SGBPdex was also fit to the cryoEM map. Unambiguous density was visible only for the first two domains of SGBPdex, and the predicted model was truncated prior to the C-terminal domain. SusCdex = purple, SusDdex = pink, GHdex = cyan and SGBPdex = green. The refinement for the heptameric complex had a global resolution of 3.1 Å. (e) Refined outputs of 3D classification viewed where each map corresponds to a unique complement or arrangement of auxiliary components (as labelled). (f) Schematic of the architecture for two apo glycan utilisomes. The levan utilisome (left) is coloured as in the main text (SusClev = green, SusDlev = gray, GHlev = blue, and SGBPlev = magenta). The equivalent schematic for the substrate-free dextran utilisome is on the right. Note the different arrangement of the GH and SGBP components relative to SusD in the levan and dextran systems.

(a) Isolated FOS density obtained from the levan utilisome dataset with active GHlev and short FOS (DP8-12)12. Density for substrate (yellow) is shown at high (left) and low (right) thresholds. (b) Isolated FOS density obtained from the utilisome structure with inactive GHlev and long FOS (DP15-25). Levan density (orange) is shown at high (left) and low (right) thresholds. Arrows indicate missing fructose branches relative to (a). At the FOS1 site, density for the putative β2,1 decoration on Frc4 is missing. Conversely, contiguous density extends beyond the previously resolved density at FOS2, with a novel β2,1 decoration on Frc5. The substrate bound at the FOS2 site follows a similar trend with the previously modelled β2,1 linked fructose side chain being much weaker with longer FOS, while additional density attributed to another β2,6 linked monomer extends the chain towards the FOS1 site. At higher threshold levels, density connects the FOS1 and FOS2 binding sites, indicating that longer FOS (~DP15) can occupy both sites simultaneously. The connecting density is weak and indicative of multiple conformations, consistent with the absence of any contacts from SusClev to this segment. These data confirm that the transporter has considerable substrate binding promiscuity and that, as suggested previously, relatively long FOS (~15 DP) can be accommodated12. FOS models shown are from the original X-ray crystal structure of the SusCDlev complex determined in the presence of short FOS (DP6-12)12. (c) Cryo-EM structure of the inactive GHlev with FOS bound (blue) superposed with the two crystal structures (7ZNR and 7ZNS; orange, grey). (d,e) Comparison of FOS bound in the FOS3 (the active site) and FOS4 (secondary) binding sites of GHlev. The arrowheads point to breaks in the FOS chain in the crystal structures, possibly as a result of using a lower DP FOS for co-crystallization than for cryo-EM. Views in (d) and (e) are generated from a superposition.

(a) Titration of 1 mM defined-length FOS into 50 μM wild type SGBPlev, suggests that ~15 fructose units are required for full affinity, which is abolished by the WAWA (W297A/W359A) mutation. (b) ITC titrations of 8 mg/ml levan, inulin or dextran 500 into 50 μM SGBPlev shows its specificity for levan. (c) ITC data from titrations of GHlev variants (all indicated residues mutated to alanine in the inactive D42A GHlev background). Levan (8 mg/ml) was titrated into 50 μM of indicated GHlev variant. Data fitting assumptions are described in the methods. (d) Surface representation of the GHlev model, with FOS shown as yellow sticks. Inset are zoomed views of the FOS3 (active site) and FOS4 (secondary) binding sites, in which atomic models in cartoon representation for FOS3 are shown with side chains for aromatic residues (Y70A, W318A). For the secondary binding site these residues are W217A, F243A, Y437A.

Source data

a, Overview and b, Close up view of the SGBPlev FOS binding site. The aromatic and polar residues that likely interact with the FOS are shown as grey stick models. The cryo-EM structure of SGBPlev was aligned with selected homologue AlphaFold2-predicted models (c–f). c, Bacteroides sp. D2 SGBPlev (UniProt E5CCB3). d, Prevotella oralis ATCC 33269 SGBPlev (E7RM14). e, Flavobacterium commune SGBPlev (A0A1D9P8I4). f,F. cellulosilyticum SGBPlev (A0A4R5CJN9). FOS-binding residues equivalent to those in b are shown as grey stick models (if present). The FOS chain from the SGBPlev cryo-EM model is shown in b–f for reference (orange and red). The identity indicated in each panel corresponds only to the C-terminal levan-binding domain sequence compared to SGBPlev from B. theta. Although we could not confidently identify which SGBPlev residues form hydrogen bonds with FOS from the cryo-EM maps, binding site conservation analysis indicates that N295, T350, Q352 and N384 of SGBPlev are likely involved in FOS binding. The amino acid sequence alignment of the models shown here can be found in Supplementary Fig. 3.

This file contains Supplementary Discussion, Figs. 1–4 and References.

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White, J.B.R., Silale, A., Feasey, M. et al. Outer membrane utilisomes mediate glycan uptake in gut Bacteroidetes. Nature (2023). https://doi.org/10.1038/s41586-023-06146-w

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Received: 08 July 2022

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Published: 07 June 2023

DOI: https://doi.org/10.1038/s41586-023-06146-w

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Outer membrane utilisomes mediate glycan uptake in gut Bacteroidetes