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Asymmetric drug binding in an ATP-loaded inward-facing state of an ABC transporter

Nature Chemical Biology volume 18, pages 226–235 (2022)Cite this article

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Abstract

Substrate efflux by ATP-binding cassette (ABC) transporters, which play a major role in multidrug resistance, entails the ATP-powered interconversion between transporter intermediates. Despite recent progress in structure elucidation, a number of intermediates have yet to be visualized and mechanistically interpreted. Here, we combine cryogenic-electron microscopy (cryo-EM), double electron–electron resonance spectroscopy and molecular dynamics simulations to profile a previously unobserved intermediate of BmrCD, a heterodimeric multidrug ABC exporter from Bacillus subtilis. In our cryo-EM structure, ATP-bound BmrCD adopts an inward-facing architecture featuring two molecules of the substrate Hoechst-33342 in a striking asymmetric head-to-tail arrangement. Deletion of the extracellular domain capping the substrate-binding chamber or mutation of Hoechst-coordinating residues abrogates cooperative stimulation of ATP hydrolysis. Together, our findings support a mechanistic role for symmetry mismatch between the nucleotide binding and the transmembrane domains in the conformational cycle of ABC transporters and is of notable importance for rational design of molecules for targeted ABC transporter inhibition.

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Fig. 1: Structure of BmrCDD500Q/E592Q in complex with Hoechst-33342 and ATP.
Fig. 2: Symmetric geometry of ATP-bound NBSs in BmrCD-QQ*H/ATP.
Fig. 3: A dynamic role of the ECD in BmrCD catalysis.
Fig. 4: Asymmetric binding of Hoechst-33342 in BmrCD-QQ*H/ATP.
Fig. 5: Cooperative stimulation of ATP turnover in BmrCD by Hoechst-33342.
Fig. 6: Analysis of alterations in the substrate-binding cavity of a BmrCD model in a putative outward-open conformation.

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Data availability

Structural data presented in this study are available for download from the Protein Data Bank and EMDB under accession codes 7M33 and EMD-23641, respectively. Raw cryo-EM micrographs/videos are also freely available to download from the Electron Microscopy Public Image Archive. Additional data can be provided on request from the authors.

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Acknowledgements

This research was funded by the Division of Intramural Research of the National Heart, Lung and Blood Institute (W.Z. and J.D.F.G.), and grants from the National Institute of General Medicine Sciences and National Institute of Allergy and Infectious Disease awarded to T.M.T. (grant nos. R00 GM114245 and R01 AI156270) and H.S.M. (grant no. R01 GM128087). Computational resources were in part provided by the National Institutes of Health (NIH) HPC facility Biowulf. A portion of this research was supported by NIH grant no. U24GM129547 and performed at the PNCC at the Oregon Health Sciences University and accessed through Environmental Molecular Sciences Laboratory (grid.436923.9), a Department of Energy Office of Science User Facility sponsored by the Office of Biological and Environmental Research. We thank T. Humphries and other support staff at the PNCC for assistance with cryo-EM data collection. We thank D. Williams with assistance with cryo-EM data collection and resources supported by National Science Foundation funding (grant no. NSF1531991) awarded to the Eyring Materials Center at Arizona State University. This work was also supported by resources in the University of Arizona Imaging Cores – Life Sciences North (grant no. S10 OD011981). We thank members of the Tomasiak laboratory and D. Claxton from the Mchaourab laboratory for critical reading of this paper.

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Author notes

  1. Smriti Mishra

    Present address: St Jude Children’s Research Hospital, Memphis, TN, USA

  2. These authors contributed equally: Tarjani M. Thaker, Smriti Mishra.

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, USA

    Tarjani M. Thaker & Thomas M. Tomasiak

  2. Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA

    Smriti Mishra, Michael Mohan, Qingyu Tang & Hassane S. Mchaourab

  3. Theoretical Molecular Biophysics Laboratory, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA

    Wenchang Zhou & José D. Faraldo-Goméz

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Contributions

T.M. Tomasiak and H.S.M. conceptualized this study. T.M. Thaker, S.M, W.Z., J.D.F.G., H.S.M. and T.M. Tomasiak conducted the experimental design, investigation and analyses. T.M. Thaker performed cryo-EM sample preparation, structure determination and analysis, and the construct design. S.M. performed DEER and biochemical experiments. W.Z. and J.D.F.G. performed MD simulation and docking experiments. M.M. and Q.T. assisted with biochemical experiments performed in revision. The initial manuscript was prepared by T. M. Thaker, T. M. Tomasiak and H.S.M., with further editing by S.M. and J.D.F.G. Data presentation and visualization were conducted by T.M. Thaker and S.M. Supervision of research and funding acquisition was carried out by T.M. Tomasiak and H.S.M.

Corresponding authors

Correspondence to Hassane S. Mchaourab or Thomas M. Tomasiak.

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Peer review information Nature Chemical Biology thanks Damian Ekiert, Lars Schäfer and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Postprocessed maps corresponding to the BmrCD-QQ* ECD.

Blurred and sharpened maps of the final unmasked postprocessed map from RELION 3.142 generated in CCP-EM48 used for modeling and refinement of the ECD in BmrCD-QQ*H/ATP. Maps shown are contoured to a sigma level of 2.0 in PyMOLv2.339.

Extended Data Fig. 2 DEER decay signals for TMD/ECD spin-labeled BmrCD* variants.

a) Cartoon representations of BmrCD subdomains highlighting the position of spin-labeled cysteine pairs generated in the transmembrane domain (TMD) and the extracellular domain (ECD). DEER decay signals and distance distributions for spin-labeled pairs generated in the b) TMD and c) TMD/ECD spin-label pairs. The shaded regions in (B) and (C) represent confidence bands. Data shown are related to main text Figs. 1 and 3.

Extended Data Fig. 3 Correlative and orthogonal analysis of NBD distances in BmrCD-QQ*.

a) Cartoon representations of the cryo-EM structure of the BmrCD nucleotide binding domain (NBD) highlighting the position of spin-labeled cysteine pairs in the consensus (440/441) and degenerate (348/532) sites. b) Comparison of symmetry axis features determined using SymD60 in symmetric BmrCD (top) and asymmetric Mrp110 (bottom). The symmetry transformed structure (grey ribbon) for each model is shown superimposed to highlight the extent of asymmetry observed in each. c) Analyzed fits (left panel) of DEER decay signals (middle panel) are shown side-by-side with confidence bands (right panel) for data collected for BmrCD-QQ* samples prepared in digitonin containing buffer. Molecular dynamics distance simulations (MDDS) curves (dashed lines in (C)) calculated using the cryo-EM structure of BmrCD as a reference are shown superimposed for clarity. Lines numbered 1-4 in the consensus data and 1-3 in the degenerate data correspond to the mean peak distances summarized in Supplementary Table 4. Peak 2 in the consensus data and 3 in the degenerate data correspond to the same population based on our analysis. Data shown are related to main text Fig. 2. d) DEER decay and confidence band data for BmrCD-QQ* NBD spin label pairs collected in β-DDM buffer.

Extended Data Fig. 4 Examination of the Hoechst-33342 binding pose with MD simulations.

Data shown are for two independent MD trajectories (black, red) of a minimal construct of BmrCD-QQ* with two Hoechst molecules bound. a) Quantification of ionic and aromatic interactions between Hoechst molecule 1 (HT1) and residues in BrmC, in terms of probability distributions of the minimum distance between the ligand and each sidechain (excluding hydrogens). The only significant contact of HT1 with BrmD is also indicated. b) Same as in (A), for molecule 2 (HT2). A summary of these c) HT1 and (d) HT2 interactions together with those observed in the cryo-EM structure determined for BmrCD-QQ*H/ATP are shown in the schematics. Interacting residues are mapped onto the respective chains (BmrC in teal; BmrD in orange). Residues colored in red are observed interacting with both molecules of Hoechst-33342 in the cryo-EM structure. Residues with an asterisk were mutated in this study. Red spheres correspond to residues identified as interacting with HT1 or HT2 in both the cryo-EM structure and in the all-atom simulation. Data shown are related to main text Fig. 4.

Extended Data Fig. 5 Hoechst-33342 binding geometries in BmrCD compared to LmrP.

Cartoon representations of A) BmrCD-QQ*H/ATP and B) LmrP36 are shown with Hoechst molecules shown as spheres. C) RMSD analysis of Hoechst-33342 bound in BmrCD-QQ*H/ATP and LmrP, with Hoechst from each respective structure shown superposed to highlight diffefrences in ligand geometry.

Extended Data Fig. 6 Enzymatic activity in BmrCD* variants.

ATP turnover rates (Vmax) in C-less wild-type BmrCD (BmrCD*) and wild-type or QQ mutants of spin-labeled cysteine-pairs. Vmax data shown are the mean ± standard error (S.E.) of at least 2 biological repeats or 3-4 technical replicates. Data shown are related to main text Fig. 5.

Supplementary information

Supplementary Information

Supplementary Figs. 1–10 and Tables 1–4.

Reporting Summary

Supplementary Data 1

Supplementary Fig. 2a uncropped SDS–PAGE gel.

Supplementary Data 2

Supplementary Fig. 2b representative cryo-EM micrograph uncropped.

Supplementary Data 3

Supplementary Fig. 3a representative cryo-EM micrograph uncropped.

Supplementary Data 4

Supplementary Fig. 3b representative cryo-EM micrograph uncropped.

Supplementary Data 5

Supplementary Fig. 10 uncropped SDS–PAGE gel.

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Thaker, T.M., Mishra, S., Zhou, W. et al. Asymmetric drug binding in an ATP-loaded inward-facing state of an ABC transporter. Nat Chem Biol 18, 226–235 (2022). https://doi.org/10.1038/s41589-021-00936-x

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