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Mechanism of cyclic β-glucan export by ABC transporter Cgt of Brucella

Abstract

Polysaccharides play critical roles in bacteria, including the formation of protective capsules and biofilms and establishing specific host cell interactions. Their transport across membranes is often mediated by ATP-binding cassette (ABC) transporters, which utilize ATP to translocate diverse molecules. Cyclic β-glucans (CβGs) are critical for host interaction of the Rhizobiales, including the zoonotic pathogen Brucella. CβGs are exported into the periplasmic space by the cyclic glucan transporter (Cgt). The interaction of an ABC transporter with a polysaccharide substrate has not been visualized so far. Here we use single-particle cryoelectron microscopy to elucidate the structures of Cgt from Brucella abortus in four conformational states. The substrate-bound structure reveals an unusual binding pocket at the height of the cytoplasmic leaflet, whereas ADP-vanadate models hint at an alternative mechanism of substrate release. Our work provides insights into the translocation of large, heterogeneous substrates and sheds light on protein-polysaccharide interactions in general.

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Fig. 1: Function of Cgt.
Fig. 2: Structural models of Cgt.
Fig. 3: Substrate interaction of Cgt.
Fig. 4: Nucleotide-bound Cgt.
Fig. 5: Model for CβG translocation by Cgt.

Data availability

Atomic structure coordinates were deposited in the PDB under accession nos. 7ZO8 (CgtAPO), 7ZOA (CgtSUB), 7ZO9 (CgtVAN) and 7ZNU (CgtDET). The cryo-EM maps were deposited in the Electron Microscopy Data Bank (EDBM) under accession nos. EMD-14843 (CgtAPO), EMD-14845 (CgtSUB), EMD-14844 (CgtVAN) and EMD-14814 (CgtDET). Source data are provided with this paper.

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Acknowledgements

We thank M. Chami, K. N. Goldie, L. Kovacik and I. Mohammed from the BioEM Lab (University of Basel, Switzerland) for support during cryo-EM data collection. We thank C. Perez and G. Cebrero Acuña (University of Basel, Switzerland) for sharing their expertise in PLS generation and handling. We thank L. Siewert for help during manuscript preparation. This work was supported by the Swiss National Science Foundation (SNSF, www.snf.ch; grant no. 310030B_201273 to C.D.), SNSF National Centres of Competence in Research (NCCR) AntiResist (grant no. 180541 to C.D.), SNSF NCCR TransCure (grant no. 185544 to H.S.) and the Chinese Scholarship Council (to N.W., project no. 201709370040).

Author information

Authors and Affiliations

Authors

Contributions

J.S., D.N. and F.L. conceived the project and designed the experiments. J.S. and F.L. expressed and purified Cgt and MSP1D1 and reconstituted Cgt into lipidic nanodiscs and PLSs. J.S. prepared cryo-EM grids. J.S. and D.N. performed cryo-EM data collection and processed EM data. D.N. built, refined and validated the structures. J.S. prepared DNA constructs and A. tumefaciens strains and carried out the functional assays. N.W. carried out the MAPDI-TOF experiments and data analysis. R.Z. provided resources and supervision for the MAPDI-TOF analysis. S.J. provided cyclic glucan samples. J.S. and D.N. prepared the manuscript. C.D. and H.S. supervised the projects and participated in manuscript writing. All authors contributed to revision of the manuscript. Any correspondence should be to H.S. or C.D.

Corresponding authors

Correspondence to Henning Stahlberg or Christoph Dehio.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Structural & Molecular Biology thanks Albert Guskov, Arne Moeller and Jochen Zimmer for their contribution to the peer review of this work. Primary Handling Editor: Carolina Perdigoto, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 In vivo Cgt activity assay.

a, Growth dynamic of Atu under osmotic stress. The Δcgt mutant can be rescued with WT B. abortus cgt gene, but not with cgtE497Q. Addition of 100 mM NaCl restores growth of all strains. b, Atu Δcgt was rescued with different point mutants of Bab cgt under low osmotic stress conditions (YPL). c, Same as b, with low osmotic stress partially alleviated by the addition of 15 mM NaCl to the medium. d, Cartoon representation of the constructs used in the assay. e, Western blot analysis of cgt-3xFlag gene expression in Atu strains used in the assay. All growth curves represent mean values of 3 independent experiments (n = 3). Error bars represent standard deviation. Data for graphs a-c and uncropped images for e are available as source data. Western blot analysis was performed once (n = 1).

Source data

Extended Data Fig. 2 Purification of Cgt in LMNG and nanodiscs.

a, Gel-filtration profile (Superose 6 Increase 10/300) of Cgt in LMNG. b, Gel-filtration profile (Superose 6 Increase 10/300) of Cgt in nanodiscs formed by MSP1D1 and E. coli polar lipids. c, MALDI-TOF analysis of Cgt in nanodiscs, indicating the presence of Flag-tagged Cgt (68.7 kDa) and MSP1D1 (22 kDa) in the sample. d, SDS-PAGE of Cgt proteoliposome sample indicating the incorporation of Flag-tagged Cgt (68.7 kDa). e, Diagram describing the CβG proteoliposome uptake assay. See the Methods section for the description. Mass spectroscopy data for c and uncropped gel image for d are available as source data. SDS-PAGE analysis was performed once (n = 1).

Source data

Extended Data Fig. 3 Cryo-EM data processing workflow for nanodisc-stabilized CgtE497Q substrate-free structure (CgtAPO).

a, Representative motion-corrected cryo-EM micrograph (scale bar is 50 nm). b, representative 2D class averages. c, Angular distribution of the cryo-EM particles included in the final reconstruction. d, Single-particle cryo-EM data processing scheme using cryoSparc 3.1. e, Final 3D reconstruction colored according to local resolution. f, Fourier shell correlation curves for the final 3D reconstruction. g, TM helices of the CgtAPO model overlapping with the corresponding experimental density. Contour levels are 1.0. Cryo-EM data was obtained in a single data collection session (n = 1).

Extended Data Fig. 4 Cryo-EM data processing workflow for nanodisc-stabilized CgtE497Q with CβG substrate (CgtSUB).

a, Representative motion-corrected cryo-EM micrograph (scale bar is 50 nm). b, representative 2D class averages. c, Angular distribution of the cryo-EM particles included in the final reconstruction. d, Single-particle cryo-EM data processing scheme using cryoSparc 3.1. e, Final 3D reconstruction colored according to local resolution. f, Fourier shell correlation curves for the final 3D reconstruction. g, TM helices of the CgtSUB model overlapping with the corresponding experimental density. Contour levels are 0.5. Cryo-EM data was obtained in two data collection sessions (n = 2).

Extended Data Fig. 5 Cryo-EM data processing workflow for nanodisc-stabilized CgtWT with Mg2+, ADP, sodium orthovanadate and CβG (CgtVAN).

a, Representative motion-corrected cryo-EM micrograph (scale bar is 50 nm). b, representative 2D class averages. c, Angular distribution of the cryo-EM particles included in the final reconstruction. d, Single-particle cryo-EM data processing scheme using cryoSparc 3.1. e, Final 3D reconstruction colored according to local resolution. f, Fourier shell correlation curves for the final 3D reconstruction. g, TM helices of the CgtVAN model overlapping with the corresponding experimental density. Contour levels are 0.5. Cryo-EM data was obtained in a single data collection session (n = 1).

Source data

Extended Data Fig. 6 Cryo-EM data processing workflow for LMNG-solubilized CgtWT with Mg2+, ADP, sodium orthovanadate and CβG (CgtDET).

a, Representative motion-corrected cryo-EM micrograph (scale bar is 50 nm). b, representative 2D class averages. c, Angular distribution of the cryo-EM particles included in the final reconstruction. d, Single-particle cryo-EM data processing scheme using cryoSparc 3.1. e, Final 3D reconstruction colored according to local resolution. F, Fourier shell correlation curves for the final 3D reconstruction. g, Local defocus values (Å) for an example micrograph indicating the sample tilt. h, TM helices of the CgtVAN model overlapping with the corresponding experimental density. Contour levels are 0.4. Cryo-EM data was obtained in a single data collection session (n = 1).

Extended Data Fig. 7 Inward-facing conformations of Cgt.

a, Alignment of single monomers of CgtAPO (purple) and CgtVAN (yellow), indicating the hinging movement of TM helices 4 and 5 that allows the transition between conformational states. b, Comparison of TM5 of CgtAPO (left) and CgtVAN (right) models. Corresponding density maps are shown. Residues P234 and G244 facilitating the bending of the helix are shown in red. Contour levels are 1.9 and 1.0. c, Alignment of TM5 of Cgt with its structural homologues. Purple, Cgt TM5; blue, TM5 of MsbA (PDB:5tv4); orange, TM11 of ABCB1 (PDB:7a6f). d, Overlay of the CgtSUB cryo-EM density map with the corresponding model at three different angles. An 18-glucose CβG molecule is shown in blue/red. Residues forming the substrate-binding pocket are shown in yellow. Contour levels are 0.25. e, Alignment of Cgt sequences from different species of Rhizobiales focusing on the substrate-binding pocket area. B. abortus residues described in this study are marked with magenta. Sequences corresponding to transmembrane helices are marked in green. Alignment was generated using Geneious. f, Slice-through views of the substrate-bound models of MsbA (PDB:5tv4, left) and CgtSUB (right), indicating distinct substrate-binding mechanisms. Respective substrates are indicated in blue. Unoccupied, inner part of the Cgt central cavity is indicated with blue shading.

Extended Data Fig. 8 Nucleotide-bound conformations of Cgt.

a-c, Top view from the periplasmic side of Cgt highlighting the hydrophobic ‘isoleucine gate’ formed by residues I46 and I276. The gate is closed in the inward-facing state (CgtAPO, a) as wells as in the closed-occluded conformation (CgtVAN, b). In the inward-facing CgtDET structure (c), the gate is open, allowing access from the central cavity to the periplasmic space. d, Periplasmic side of Cgt TM helices, indicating the proposed mechanism for opening of the central cavity to the outside. Chain A shown in purple and pink; chain B shown in blue and cyan. A CβG molecule (blue-red sticks) was docked into the central cavity. Rearrangements of TMs and periplasmic loops between the CgtVAN (left) and CgtDET (right) models are indicated with arrows. The resulting opening allows the release of the substrate into the periplasmic space (grey arrow).

Extended Data Fig. 9 Cgt substrate translocation.

a, Side view of the central cavity of the CgtSUB (left) and CgtDET (right) models, indicating the CβG binding pocket residues (purple) and the free space in the central cavity (blue shading). Residues pointing towards the center of the inner cavity in both models that were targeted by Trp mutagenesis are shown in yellow. b, View of the inner part of the central cavity from the periplasmic space in CgtSUB (top) and CgtDET (bottom) models. Residues mutated to Trp are shown in yellow. c, d, In-vivo Cgt activity assay indicating the ability of different Trp mutants of Cgt to restore growth of Atu Δcgt under osmotic stress. Both growth curves (c) and a single 20 h time point (d) are shown. Growth curves represent mean values of 3 independent experiments (n = 3). Dots represent mean values of independent experiments (n = 3). Error bars represent standard deviation. Data for graphs c-d are available as source data.

Source data

Supplementary information

Reporting Summary

Supplementary Table 1

List of primers used in the study.

Supplementary Table 2

List of plasmids used in the study.

Supplementary Video 1

Membrane topology of Cgt.

Supplementary Video 2

Substrate-induced conformational changes of Cgt.

Supplementary Video 3

Experimental density of the cyclic glucan within the binding pocket.

Supplementary Video 4

The substrate-binding pocket of Cgt.

Supplementary Video 5

Dimerization of NBDs and closing of the Cgt central cavity.

Supplementary Video 6

Transition between the two nucleotide-bound structures of Cgt.

Supplementary Video 7

The transporting cycle of Cgt.

Source data

Source Data Fig. 1

Bacterial growth data for Fig. 1b, ATPase activity assay data for Fig. 1c and MS results data for Fig. 1d,e.

Source Data Fig. 3

Bacterial growth data for Fig. 3e.

Source Data Extended Data Fig. 1

Bacterial growth data for Extended Data Fig. 1a–c.

Source Data Extended Data Fig. 1

Uncropped western blot images, including luminescence images as well as pictures showing Mr markers.

Source Data Extended Data Fig. 2

MS results data for Extended Data Fig. 2c.

Source Data Extended Data Fig. 2

Uncropped SDS–PAGE gel image.

Source Data Extended Data Fig. 9

Bacteria growth data for Extended Data Fig. 9c,d.

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Sedzicki, J., Ni, D., Lehmann, F. et al. Mechanism of cyclic β-glucan export by ABC transporter Cgt of Brucella. Nat Struct Mol Biol 29, 1170–1177 (2022). https://doi.org/10.1038/s41594-022-00868-7

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