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Structural basis for nutrient acquisition by dominant members of the human gut microbiota

Nature volume 541, pages 407–411 (2017)Cite this article

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Abstract

The human large intestine is populated by a high density of microorganisms, collectively termed the colonic microbiota1, which has an important role in human health and nutrition2. The survival of microbiota members from the dominant Gram-negative phylum Bacteroidetes depends on their ability to degrade dietary glycans that cannot be metabolized by the host3. The genes encoding proteins involved in the degradation of specific glycans are organized into co-regulated polysaccharide utilization loci4,5,6,7,8, with the archetypal locus sus (for starch utilisation system) encoding seven proteins, SusA–SusG8,9,10. Glycan degradation mainly occurs intracellularly and depends on the import of oligosaccharides by an outer membrane protein complex composed of an extracellular SusD-like lipoprotein and an integral membrane SusC-like TonB-dependent transporter4,5,6,7,11,12,13. The presence of the partner SusD-like lipoprotein is the major feature that distinguishes SusC-like proteins from previously characterized TonB-dependent transporters. Many sequenced gut Bacteroides spp. encode over 100 SusCD pairs, of which the majority have unknown functions and substrate specificities3,8,14,15. The mechanism by which extracellular substrate binding by SusD proteins is coupled to outer membrane passage through their cognate SusC transporter is unknown. Here we present X-ray crystal structures of two functionally distinct SusCD complexes purified from Bacteroides thetaiotaomicron and derive a general model for substrate translocation. The SusC transporters form homodimers, with each β-barrel protomer tightly capped by SusD. Ligands are bound at the SusC–SusD interface in a large solvent-excluded cavity. Molecular dynamics simulations and single-channel electrophysiology reveal a ‘pedal bin’ mechanism, in which SusD moves away from SusC in a hinge-like fashion in the absence of ligand to expose the substrate-binding site to the extracellular milieu. These data provide mechanistic insights into outer membrane nutrient import by members of the microbiota, an area of major importance for understanding human–microbiota symbiosis.

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Figure 1: Overall architecture of SusCD complexes.
Figure 2: The SusC–SusD interaction involves the ligand-binding face of SusD.
Figure 3: The SusCD ligand-binding site is conserved.
Figure 4: SusCD complexes transport their substrates through a pedal bin mechanism.

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Acknowledgements

We would like to thank J. Gray for B. theta outer membrane protein identification, R. Lewis for critical reading of the manuscript and S. Buchanan for the pET9 expression vector. We thank O. Davies for help with SEC–MALS analysis. We are also indebted to the staff at beamlines I24, I04 and I03 of the Diamond Light Source UK for beam time (proposal mx9948) and assistance with data collection. A.J.G. acknowledges support from the Barbour Foundation. S.B.P. was funded by EU FP7-PEOPLE-2013-ITN Translocation network Nr. 607694. The research of K.R.P., U.K., M.W. and B.v.d.B. has received support from the Innovative Medicines Initiatives Joint Undertaking under Grant Agreement No. 115525, resources which are composed of financial contributions from the European Union’s seventh framework programme (FP7/2007-2013) and European Federation of Pharmaceutical Industries and Associations companies in-kind contribution.

Author information

Author notes

  1. Amy J. Glenwright and Karunakar R. Pothula: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK

    Amy J. Glenwright, Arnaud Baslé, Susan J. Firbank, Hongjun Zheng, David N. Bolam & Bert van den Berg

  2. Department of Physics & Earth Sciences, Jacobs University Bremen, Bremen, 28759, Germany

    Karunakar R. Pothula & Ulrich Kleinekathöfer

  3. Department of Life Sciences & Chemistry, Jacobs University Bremen, Bremen, 28759, Germany

    Satya P. Bhamidimarri & Mathias Winterhalter

  4. Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QZ, UK

    Dror S. Chorev & Carol V. Robinson

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Contributions

A.J.G. expressed, purified and crystallized proteins and carried out western blot analysis. K.R.P. and U.K. performed molecular dynamics simulations. S.P.B. and M.W. performed and supervised electrophysiology experiments, respectively. D.S.C. and C.V.R. carried out mass-spectrometry analyses. A.B. collected crystallography data and managed the Newcastle Structural Laboratory. H.Z. expressed and purified BT1762 and performed ITC measurements. S.J.F. and A.B. determined the structure of BT1762. D.N.B. designed research and assisted in writing the paper. B.v.d.B. crystallized proteins and solved crystal structures, designed research and wrote the paper.

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Correspondence to Bert van den Berg.

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Nature thanks B. Henrissat, S. White and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 High abundance of SusCD complexes in B. theta outer membrane.

a, SDS–PAGE gel of total outer membranes from E. coli (lanes 1, 2, 5, 6) and B. theta (lanes 3, 4, 7, 8). Rich medium, even lanes; minimal medium, odd lanes. Each lane contains approximately 10 μg protein. Samples 5–8 were boiled. ‘P’ denotes E. coli trimeric porins OmpF/C, which migrate at their monomeric molecular weights (around 35 kDa) only after boiling. Note the relative lack of small-molecule outer membrane diffusion channels (around 30–50 kDa) in B. theta (lanes 7, 8) and the low levels of large outer membrane proteins including TBDTs (70–120 kDa) in E. coli (lanes 5, 6). Purified BT2261–64 complex is shown in lanes 9 (non-boiled) and 10 (boiled). b, Representative ion-exchange chromatogram from three separate experiments of B. theta total outer membrane proteins separated on Resource-Q (6 ml; pH 7.5) after extraction in LDAO (Methods). Peaks A and B were further purified by gel filtration. c, SDS–PAGE gel of purified SusCD complexes from peaks A and B. Numbered gel bands were excised and subjected to identification by mass spectrometry. d, SDS–PAGE gel of purified BT1762–BT1763 complex before (asterisk) and after boiling. The SusCD complexes are highly stable and remain intact in 2% SDS.

Extended Data Figure 2 X-ray crystal structures of the small lipoproteins BT2261 and BT2262.

a, Stereo cartoon of BT2261 within the BT2261–64 complex with rainbow colouring (blue; N terminus). BT2261 is O-glycosylated at Ser117, consistent with the presence of the Bacteroidetes glycosylation motif D-(S/T)-(A/L/V/I/M/T)51. Ser117 is shown as a stick model. Fo − Fc density within 20 Å of Ser117 is shown as a green mesh contoured at 3.0σ. Three to four sugar moieties can be observed bound to Ser117. b, Stereo cartoon of soluble BT2262 with rainbow colouring. The protein consists of an N-terminal Ig-like domain and a C-terminal eight-stranded β-barrel. The functions of BT2261 and BT2262 are not clear, but both contain a small C-terminal eight-stranded β-barrel that displays structural similarity to lipid binding domains as judged by DALI52. For BT2262, only one copy with a poorly ordered C-terminal domain is visible in the triclinic structure. Analogous to BT2263, the N-terminal segments of BT2261/BT2262 that lead to the lipid anchors on the N-terminal cysteine residues are visible in the electron density; they are closely associated with SusC and do not appear to be flexible. Structures were determined using data obtained from a single crystal in each case.

Extended Data Figure 3 The oligomeric nature of SusCD complexes is not a consequence of crystal packing.

a–c, Cartoon side views of BT2261–64 complexes rotated by 90° for space groups P1 (a), P212121 (b) and SeMet P21 (c). d, Cartoon side view of BT1762–BT1763 (P212121). The protein backbones are coloured on the same scale by their B-factors (blue; 20 Å2, red; 130 Å2). The grey bars indicate the hydrophobic phase of the outer membrane. Structures were determined using data obtained from a single crystal in each case.

Extended Data Figure 4 BT2261–64 and BT1762–BT1763 form oligomeric complexes.

a, Mass spectrum of BT2261–64 shows two prominent masses corresponding to an octamer and a ligand bound octamer in m/z = 12,000–15,000. b, Mass spectrum of BT1762–BT1763 indicates that these two proteins form dimers and tetramers. The numbers in parentheses on the right are the theoretical masses. c, Analytical gel filtration chromatography of BT2261–64 (blue) and BT1762–BT1763 (green). For comparison, samples were run for soluble horse spleen ferritin (440 kDa; red) and for the membrane protein ammonium transporter Mep2 from Candida albicans (160 kDa; black). The following buffer was specifically for this experiment: 10 mM HEPES/100 mM NaCl/0.12% DM pH 7.5. Column: Superdex-200 Increase 10/300 GL. d, SEC–MALS analysis of BT1762–BT1763. Light scattering (LS) and differential refractive index (dRI) are plotted alongside the fitted total protein-detergent complex molecular weight (diamonds), and constituent protein (pluses) and detergent (crosses) molecular weights, across each peak. BT1762–BT1763 eluted as two species of 499 kDa (protein component 319 kDa, corresponding to a SusCD dimer) and 269 kDa (protein component 214 kDa). Chromatograms shown are from single experiments.

Extended Data Figure 5 Unbiased electron density for the bound ligand in BT2261–64.

Stereo views of simulated annealing omit maps using a starting temperature of 1000 K. a, 2Fo − Fc maps contoured at 1.5σ; carve, 2. b, Fo − Fc map contoured at 3.0σ; carve, 2. Selected residues contacting ligand are shown (yellow; BT2264/SusC, magenta, BT2263/SusD). Density for at least six amino acid side chains is present (denoted by an asterisk in the 2Fo − Fc map). c, Interaction table showing hydrogen-bond distances between the putative peptide ligand backbone and residues in BT2263 and BT2264.

Extended Data Figure 6 Molecular dynamics simulations for BT2261–64.

a, Plots of BT2264 (SusC) Cα r.m.s.d. versus simulation time for holo- and apo-complexes. b, c, Plots of BT2263 (SusD) Cα r.m.s.d. versus simulation time for holo and apo simulations, relative to the starting conformation (b) and after SusD superposition (c). d, e, Plots showing the number of hydrogen bonds between SusC and SusD versus simulation time (d) and between holo-SusCD and the modelled peptide (e). Simulations are numbered as follows: sim1–3, apo-BT2263–BT2264 (dimer); sim7–9, apo-BT2261–64 (tetramer); sim13, apo-(BT2261–64) × 2 (octamer); sim4–6, holo-BT2263–BT2264; sim10–12, holo-BT2261–64; sim14, holo-(BT2261–64) × 2. With the exception of those of the octamer (owing to its very large size), the simulations were repeated three times with different initial atomic velocities to allow sampling in order to obtain a measure of the possible spread in results.

Extended Data Figure 7 Dynamics of the bound peptide during molecular dynamics simulations.

a–c, Side views (left panels) and top views showing the bound peptide in the BT2263–BT2264 dimers (a), BT2261–64 tetramers (b) and the (BT2261–64) × 2 octamer (c). For clarity, only one final conformation for BT2264 (SusC) is shown together with the starting conformation of the peptide (green) and the final peptide conformations after 500 ns of simulation (red). For orientation purposes, the assigned N termini of the peptides are coloured blue.

Extended Data Figure 8 Molecular dynamics simulation root-mean-square-fluctuation analyses.

a, Cα root-mean-square-fluctuation values for SusC in holo- and apo-complexes with the conformational fluctuations of the hinge loop L7 highlighted separately. b, Cα root-mean-square-fluctuation values for SusD in apo and holo simulations. Simulations are numbered as in Extended Data Fig. 6: sim1–3, apo-BT2263–BT2264 (dimer); sim7–9, apo-BT2261–64 (tetramer); sim13, apo-(BT2261–64) × 2 (octamer); sim4–6, holo-BT2263–BT2264; sim10–12, holo-BT2261–64; sim14, holo-(BT2261–64) × 2.

Extended Data Figure 9 Structure of the BT2261–64 apo-octamer after 500 ns of molecular dynamics simulation (sim13), demonstrating the independent bin opening of the two SusCD hubs.

a, Views from the plane of the membrane rotated by 90°. b, View from the extracellular side. For clarity, the SusC and SusD subunits are shown in different colours (yellow and orange for SusC/BT2264, magenta and red for SusD/BT2263). BT2261 and BT2262 are shown in green and blue, respectively. c, Side view of the opened SusCD monomer highlighting the remaining interactions between SusC (yellow) and SusD (magenta) mediated by the SusC hinge loop L7 and loop L8.

Extended Data Figure 10 ITC analysis of levan binding for recombinant BT1762 SusD-like wild-type and mutants.

a, Titration curves from single experiments. Upper panels show raw injection heats of ligand (levan) into protein, lower panels show the integrated binding heats fit to a single set of sites binding model to determine Ka for all proteins except reduced wild type (10 mM TCEP), W85A and C298A mutants that display no binding. Levan stock solution was between 0.5–2% (w/v) and protein ranged from 50–60 μM. b, Levan affinity of recombinant BT1762 SusD-like wild type and mutant proteins determined by ITC. Ka values shown are averages and standard deviations from at least two independent titrations. Residue numbering is that of the mature protein (first residue Cys1).

Supplementary information

Supplementary Information

This file contains X-ray Crystallographic Tables showing data collection and refinement statistics for BT2261-64 (Supplementary Table 1), soluble proteins (Supplementary Table 2) and BT1762-63 (Supplementary Table 3). (PDF 50 kb)

Pedal bin opening of BT2263-64 (dimer)

Side-by-side movies showing 500 ns unbiased MD simulations of BT2263-64 in the presence (left; sim6) and absence (right; sim3) of the bound peptide ligand, shown as green spheres. BT2263 (SusD) is coloured magenta, BT2264 (SusC) yellow. The plug domain of SusC is coloured dark blue. (MOV 15918 kb)

Pedal bin opening of BT2261-64 (tetramer)

Side-by-side movies showing 500 ns unbiased MD simulations of BT2261-64 in the presence (left; sim11) and absence (right; sim8) of the bound peptide ligand, shown as green spheres. BT2263 (SusD) is coloured green, BT2264 (SusC) cyan. The small lipoproteins BT2261 and BT2262 are coloured grey and dark blue, respectively. (MOV 17469 kb)

Pedal bin opening of (BT2261-64)x 2 (octamer)

Side-by-side movies showing 500 ns unbiased MD simulations of 2 x (BT2261-64) in the presence (left; sim14) and absence (right; sim13) of the bound peptide ligand, shown as green spheres. BT2263 (SusD) is coloured green, BT2264 (SusC) cyan. The small lipoproteins BT2261 and BT2262 are coloured grey and dark blue, respectively. (MOV 19802 kb)

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Glenwright, A., Pothula, K., Bhamidimarri, S. et al. Structural basis for nutrient acquisition by dominant members of the human gut microbiota. Nature 541, 407–411 (2017). https://doi.org/10.1038/nature20828

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