Abstract
Bedaquiline (BDQ), a first-in-class diarylquinoline anti-tuberculosis drug, and its analogue, TBAJ-587, prevent the growth and proliferation of Mycobacterium tuberculosis by inhibiting ATP synthase1,2. However, BDQ also inhibits human ATP synthase3. At present, how these compounds interact with either M. tuberculosis ATP synthase or human ATP synthase is unclear. Here we present cryogenic electron microscopy structures of M. tuberculosis ATP synthase with and without BDQ and TBAJ-587 bound, and human ATP synthase bound to BDQ. The two inhibitors interact with subunit a and the c-ring at the leading site, c-only sites and lagging site in M. tuberculosis ATP synthase, showing that BDQ and TBAJ-587 have similar modes of action. The quinolinyl and dimethylamino units of the compounds make extensive contacts with the protein. The structure of human ATP synthase in complex with BDQ reveals that the BDQ-binding site is similar to that observed for the leading site in M. tuberculosis ATP synthase, and that the quinolinyl unit also interacts extensively with the human enzyme. This study will improve researchers’ understanding of the similarities and differences between human ATP synthase and M. tuberculosis ATP synthase in terms of the mode of BDQ binding, and will allow the rational design of novel diarylquinolines as anti-tuberculosis drugs.
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Data availability
Electron microscopy maps and atomic coordinates have been deposited in the Electron Microscopy Data Bank (https://www.ebi.ac.uk/pdbe/emdb) and the PDB (https://www.rcsb.org/), respectively, under the following accession codes: EMD-35911 and PDB 8J0T for the ATP synthase in the apo form; EMD-35983 and PDB 8J58 for the Fo of ATP synthase in the apo form; EMD-36028 for the F1 of ATP synthase in the apo form; EMD-36015 for the peripheral stalk of ATP synthase in the apo form; EMD-35909 and PDB 8J0S for the ATP synthase in complex with BDQ; EMD-35982 and PDB 8J57 for the Fo of ATP synthase in complex with BDQ; EMD-36031 for the F1 of ATP synthase in complex with BDQ; EMD-36017 for the peripheral stalk of ATP synthase in complex with BDQ; EMD-36589 and PDB 8JR0 for the ATP synthase in complex with TBAJ-587; EMD-36590 and PDB 8JR1 for the Fo of ATP synthase in complex with TBAJ-587; EMD-36631 for the F1 of ATP synthase in complex with TBAJ-587; EMD-36632 for the peripheral stalk of ATP synthase in complex with TBAJ-587; EMD-37251 and PDB 8KI3 for the human ATP synthase in complex with BDQ; EMD-37243 and PDB 8KHF for the Fo of human ATP synthase in complex with BDQ; EMD-37244 for the F1 of human ATP synthase in complex with BDQ; and EMD-37245 for the peripheral stalk of human ATP synthase in complex with BDQ. Source data are provided with this paper.
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Acknowledgements
We thank the Advanced Bio-imaging Technology Platform of Guangzhou Laboratory and the Bio-Electron Microscopy Facility of ShanghaiTech University for technical assistance. This work was funded by the National Key R&D Program of China (grant 2022YFA1305900 to H.G.), grants from the National Natural Science Foundation of China (82222042 and 32100976 to H.G.; 32394010 to Z.R.), the Tianjin Natural Science Foundation (grant 23JCZDJC00760 to H.G.), the Young Elite Scientists Sponsorship Program by China Association for Science and Technology (grant 2023QNRC001 to F.L.) and the Shanghai Rising-Star Program (grant 23QA1406400 to Y.G.).
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H.G. conceived, initiated, coordinated and supervised the research. Yuying Zhang and Y.L. purified and performed characterization of the M. tuberculosis ATP synthase. F.L., Y.G., Yuying Zhang and Y.L. collected and processed cryo-EM data and built the structure model. H.G., Y.L., Yuying Zhang, S.Z., T.R., Yue Zhang, J.X., Z.F., L.Y., Z.Z., K.S., J.W., Y.P., L.L., H.C., Y.G., F.L., L.W.G. and Z.R. analysed the structure and discussed the results. The manuscript was written by H.G., Y.L., Yuying Zhang and L.G.
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Extended data figures and tables
Extended Data Fig. 1 Purification and characterization of M. tuberculosis ATP synthase.
a, Steps for the construction of a M. smegmatis strain expressing M. tuberculosis ATP synthase. b, Superose 6 increase size-exclusion chromatography elution profile for the M. tuberculosis ATP synthase. c, The analysis of M. tuberculosis ATP synthase components by BN–PAGE. For gel source data, see Supplementary Fig. 1a. The experiment was repeated independently 3 times with similar results. d, Peak fractions assessed by SDS–PAGE after gel filtration. Band identities were identified by mass spectrometry. For gel source data, see Supplementary Fig. 1b. The experiment was repeated independently 3 times with similar results. e, ATP hydrolysis activity for wild-type M. tuberculosis ATP synthase and the α-truncated mutant. The value depicted by each bar represents the mean from n = 3 separate assays. The errors are represented as standard deviations from the mean. f, Effects of BDQ (circles) and TBAJ-587 (triangles) on ATP hydrolysis by ATP synthase with truncated α-subunits. The mean values are shown based on n = 3 separate assays. Data are presented as mean values ± s.d. Full activity is defined as the identical assay but with no drug added.
Extended Data Fig. 2 Cryo-EM data processing of M. tuberculosis ATP synthase in the apo form and human ATP synthase in complex with BDQ.
a, M. tuberculosis ATP synthase in the apo form. b, Human ATP synthase in complex with BDQ. The BDQ- and TBAJ-587-bound forms have the same processing flow as for the M. tuberculosis ATP synthase in the apo form.
Extended Data Fig. 3 GSFSC curves and 3D FSC histograms for M. tuberculosis ATP synthase.
a–d, GSFSC curves and 3D FSC histograms for M. tuberculosis ATP synthase in the apo form (a), BDQ-bound form (b) and TBAJ-587-bound form (c) and BDQ-bound human ATP synthase (d).
Extended Data Fig. 4 Examples of polypeptide cryo-EM density from BDQ-bound M. tuberculosis and BDQ-bound human ATP synthase.
a, BDQ-bound M. tuberculosis. b, BDQ-bound human ATP synthase. The maps are contoured at a threshold of 5.6.
Extended Data Fig. 5 Structural and sequence comparison between M. tuberculosis and M. smegmatis ATP synthases.
a, Structural comparison. b, Sequence alignment statistics. c, Differences in sequence between M. tuberculosis and M. smegmatis ATP synthases are identified by the wheat-coloured spheres.
Extended Data Fig. 6 Inhibitors that target M. tuberculosis ATP synthase.
Six inhibitors of ATP synthase and their target sites and sequences. The coloured symbols indicate the residues that interact with the corresponding inhibitor.
Extended Data Fig. 7 BDQ resistance mutations in M. tuberculosis ATP synthase.
The mutations reported in the clinical isolates of M. tuberculosis are shown as stick models.
Extended Data Fig. 8 Interactions of M. smegmatis ATP synthase and BDQ.
a, Structural comparison between M. tuberculosis ATP synthase subunits a/c and that of M. smegmatis. b, The three binding sites for TBAJ-587 to M. smegmatis ATP synthase. c, Interactions between BDQ and M. smegmatis ATP synthase in three binding sites. BDQ and the surrounding residues within 5.0 Å are shown in sticks. d, Two-dimensional plot of the BDQ with M. smegmatis ATP synthase interactions. Hydrogen bonds are represented by the black dashed lines.
Extended Data Fig. 9 Inhibition of human ATP synthase by BDQ and TBAJ-587.
a, Inhibition of human ATP synthase by BDQ and TBAJ-587. The value depicted by each bar represents the mean from n = 3 separate assays, and the error bars represent the standard deviation from the mean. Full activity is defined when only DMSO is added. b, Local cryo-EM maps for the human ATP synthase with/without BDQ and TBAJ-587. The corresponding density for BDQ is shown in red, but no density for TBAJ-587 was observed. The maps for apo, BDQ- and TBAJ-587-bound human ATP synthase are display at 0.141, 0.109 and 0.139 threshold, respectively. c, Surface plasmon resonance analysis of binding affinity between human ATP synthase and BDQ/TBAJ-587. The KD values of the human ATP synthase with BDQ and TBAJ-587 calculated by using a 1 : 1 binding model.
Extended Data Fig. 10 Comparison of the binding of BDQ in the leading site in three ATP synthases.
a, Sequence alignment of the residues surrounding the BDQ-binding site of M. tuberculosis, M. smegmatis and human ATP synthase. The yellow shading indicates the residues that interact with BDQ. b,c, Two-dimensional plot showing the interacting residues in M. tuberculosis and M. smegmatis (b) and M. tuberculosis and human (c). Conserved residues are circled in red. Hydrogen bonds are represented by the black dashed lines.
Supplementary information
Supplementary Information
This file contains Supplementary Figure 1 and Supplementary Tables 1–8
Supplementary Video 1
Binding of BDQ to the M. tuberculosis ATP synthase. The video shows interpolation between the conformation of the ATP synthase in the BDQ-free and BDQ-bound states.
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Zhang, Y., Lai, Y., Zhou, S. et al. Inhibition of M. tuberculosis and human ATP synthase by BDQ and TBAJ-587. Nature 631, 409–414 (2024). https://doi.org/10.1038/s41586-024-07605-8
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DOI: https://doi.org/10.1038/s41586-024-07605-8
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