Abstract
Tuberculosis—the world’s leading cause of death by infectious disease—is increasingly resistant to current first-line antibiotics1. The bacterium Mycobacterium tuberculosis (which causes tuberculosis) can survive low-energy conditions, allowing infections to remain dormant and decreasing their susceptibility to many antibiotics2. Bedaquiline was developed in 2005 from a lead compound identified in a phenotypic screen against Mycobacterium smegmatis3. This drug can sterilize even latent M. tuberculosis infections4 and has become a cornerstone of treatment for multidrug-resistant and extensively drug-resistant tuberculosis1,5,6. Bedaquiline targets the mycobacterial ATP synthase3, which is an essential enzyme in the obligate aerobic Mycobacterium genus3,7, but how it binds the intact enzyme is unknown. Here we determined cryo-electron microscopy structures of M. smegmatis ATP synthase alone and in complex with bedaquiline. The drug-free structure suggests that hook-like extensions from the α-subunits prevent the enzyme from running in reverse, inhibiting ATP hydrolysis and preserving energy in hypoxic conditions. Bedaquiline binding induces large conformational changes in the ATP synthase, creating tight binding pockets at the interface of subunits a and c that explain the potency of this drug as an antibiotic for tuberculosis.
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Data availability
Cryo-EM maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-22311 to EMD-22322. Atomic models have been deposited in the Protein Data Bank under accession numbers 7JG5, 7JG6, 7JG7, 7JG8, 7JG9, 7JGA, 7JGB and 7JGC. Strains and plasmids are available from the corresponding author upon reasonable request.
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Acknowledgements
We thank V. Kanelis for suggestions on the manuscript. H.G. was supported by an Ontario Graduate Scholarship for International Students. G.M.C. was supported by a W. P. Caven Memorial Fellowship. J.L.R. was supported by the Canada Research Chairs programme. This work was supported by Canadian Institutes of Health Research grant PJT162186 (J.L.R.) and PJT156261 (J.L.). Cryo-EM data were collected at the Toronto High-Resolution High-Throughput cryo-EM facility, supported by the Canada Foundation for Innovation and Ontario Research Fund.
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Contributions
J.L.R. conceived the project and supervised the research. J.L. advised on M. smegmatis cell culture and chromosomal tagging strategies. S.A.B. prepared the 3×FLAG ORBIT payload plasmid and an M. smegmatis strain used in the initial stages of the project, with help from J.M. G.M.C. prepared the M. smegmatis strains used for structure determination and enzyme assays. G.M.C. and H.G. purified the protein, H.G. performed the cryo-EM and image analysis, and G.M.C. and H.G. constructed the atomic models. J.L.R., H.G. and G.M.C. wrote the manuscript and prepared figures with input from the other authors.
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Extended data figures and tables
Extended Data Fig. 1 Workflow for cryo-EM image analysis for BDQ-free, BDQ-saturated and -washed M. smegmatis ATP synthase.
a, b, Example micrograph (a) and 2D class average images (b) for BDQ-saturated M. smegmatis ATP synthase. Example particles are circled in white. We collected 7,691 movies (including 1,435,679 particle images) for the drug-free condition, 7,589 movies (including 1,825,672 particle images) for the BDQ-saturated condition and 4,962 movies (including 1,865,961 particle images) for the BDQ-washed condition. c, d, Workflow for obtaining maps of the three rotational states (c) and FO region (d).
Extended Data Fig. 2 Cryo-EM map validation.
a–d, Corrected Fourier shell correlation curves after gold-standard refinement, orientation distribution plots and local resolution maps are shown for maps of the entire complex from the BDQ-free dataset (a), BDQ-saturated dataset (b) and BDQ-washed dataset (c), as well as FO-region maps from focused refinement (d).
Extended Data Fig. 3 Examples of model-in-map fit to demonstrate map quality.
a, b, Examples are shown from each subunit in the BDQ-free model (a) and BDQ-saturated model (b).
Extended Data Fig. 4 Comparison of subunits a and b–δ from different organisms.
a, The Bacillus PS3 subunit a is in grey and the M. smegmatis subunit a is in green. b, The E. coli subunit a is in purple and the M. smegmatis subunit a is in green. c, The N-terminal domain of the δ region of the b–δ fusion protein (right side, top) is substantially larger than the N-terminal domain of Bacillus PS3 δ-subunit (right side, middle), being composed of about 270 residues instead of about 170 residues. The extra sequence contributes 5 additional α-helices to the 6 found in the Bacillus PS3 δ-subunit, giving a total of 11 α-helices in the N-terminal domain of b–δ (Fig. 1b, bottom). Mycobacterium smegmatis b–δ and Bacillus PS3 δ also form different interactions with the N-terminal sequences of the α-subunits that help to attach the peripheral stalk to F1 region (right side, red).
Extended Data Fig. 5 Two conformations of the α-extension are seen in rotational state 2.
Density for the α-extension in its predominant conformation is coloured pink, and the alternative conformation is coloured red.
Extended Data Fig. 6 The BDQ-washed structures are in the BDQ-saturated conformation.
Fit of the BDQ-saturated protein models in the BDQ-washed maps shows that the two preparations adopt the same conformation.
Supplementary information
Supplementary Table 1
Cryo-EM data collection, refinement and validation statistics. This file contains Supplementary Tables 2-4 and Supplementary Figures 1-2.
Video 1
Release of the inhibitory α extension during ATP synthesis. The video shows interpolation between the different rotational states of the enzyme, including the alternative conformation of rotational state 2, which suggests the sequence of events that occur during release of the α-extension during ATP synthesis and binding of the α-extension to subunit γ to inhibit ATP hydrolysis. The video may be viewed as a loop.
Video 2
Binding of BDQ to the mycobacterial ATP synthase. The video shows interpolation between the conformation of the ATP synthase in the BDQ-free and BDQ-bound states. BDQ in the leading, c-only, and lagging binding sites are coloured pink, yellow, and blue, respectively. Phe69 in subunit c and Phe221 in subunit a, which undergo large conformational changes upon drug binding, are shown as space-filling models. The video may be viewed as a loop.
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Guo, H., Courbon, G.M., Bueler, S.A. et al. Structure of mycobacterial ATP synthase bound to the tuberculosis drug bedaquiline. Nature 589, 143–147 (2021). https://doi.org/10.1038/s41586-020-3004-3
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DOI: https://doi.org/10.1038/s41586-020-3004-3
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