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Single-molecule probing of the conformational homogeneity of the ABC transporter BtuCD

Nature Chemical Biology volume 14, pages 715–722 (2018)Cite this article

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Abstract

ATP-binding cassette (ABC) transporters use the energy of ATP hydrolysis to move molecules through cellular membranes. They are directly linked to human diseases, cancer multidrug resistance, and bacterial virulence. Very little is known of the conformational dynamics of ABC transporters, especially at the single-molecule level. Here, we combine single-molecule spectroscopy and a novel molecular simulation approach to investigate the conformational dynamics of the ABC transporter BtuCD. We observe a single dominant population of molecules in each step of the transport cycle and tight coupling between conformational transitions and ligand binding. We uncover transient conformational changes that allow substrate to enter the transporter. This is followed by a ‘squeezing’ motion propagating from the extracellular to the intracellular side of the translocation cavity. This coordinated sequence of events provides a mechanism for the unidirectional transport of vitamin B12 by BtuCD.

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Fig. 1: smFRET experimental design.
Fig. 2: Conformational distributions and conformational stability of the NBDs.
Fig. 3: Conformational dynamics of the NBDs.
Fig. 4: Conformational distributions of the TMDs and differences between the membrane and detergent environments.
Fig. 5: Conformational dynamics of the TMDs.
Fig. 6: Molecular simulations and proposed mechanism of BtuCD.

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Acknowledgements

This work is in honor and memory of Yongfang Zhao.The authors thank B. Poolman and A. Meller for fruitful discussions. The plasmid for Cys-less BtuCD was a kind gift from K. Locher (ETH Zurich). This work was supported by grants from NATO Science for Peace and Security Program (SPS Project G4622, O.L., N.L.L., J.R., T.H., B.A., B.A.F., N.B.T., and G.M.), the Israeli Academy of Sciences (O.L., N.L.L.), TUBITAK (The Scientific and Technological Research Council of Turkey) under the grant no 115M418 (T.H., B.A., B.A.F.), the Rappaport Family Institute for biomedical research (O.L., N.L.L., J.R.), the Ministry of Science and Technology (China) “973” Project grant 2014CB910400 (M.Y., J.Z.), the National Natural Science Foundation of China (31522016) (M.Y. and Y.Z.), and the Merieux research foundation (O.L., N.L.L.).

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Author notes

  1. These authors contributed equally: Min Yang, Nurit Livnat Lavanon.

Authors and Affiliations

  1. National Laboratory of Macromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Min Yang & Yongfang Zhao

  2. Department of Biochemistry and the Rappaport Institute for Medical Sciences, Faculty of Medicine, The Technion-Israel Institute of Technology, Haifa, Israel

    Nurit Livnat Levanon, Jessica Rose & Oded Lewinson

  3. Department of Chemical Engineering and Polymer Research Center, Bogazici University, Istanbul, Turkey

    Burçin Acar, Burcu Aykac Fas & Turkan Haliloglu

  4. Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel

    Gal Masrati & Nir Ben-Tal

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Contributions

M.Y. performed the smFRET measurements; N.L.L. constructed the mutants and conducted the functional assays; B.A., B.A.F., and J.R. conducted the molecular simulations; G.M. conducted the HOLE analysis; and all authors analyzed data. Y.Z. and O.L. conceptualized the project; Y.Z., O.L., N.B.-T., and T.H. directed the project. O.L. wrote the manuscript with assistance from all authors.

Corresponding authors

Correspondence to Nir Ben-Tal, Turkan Haliloglu or Oded Lewinson.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Tables 1–3

Reporting Summary

Supplementary Video 1

The gradual formation of the ATP-binding site

Supplementary Video 2

Conformational changes of the TMDs

Supplementary Video 3

Conformational changes of the complete transporter

Supplementary Video 4

Opening of the periplasmic gate

Supplementary Video 5

Upward movement of the periplasmic gate

Supplementary Video 6

Remodeling of the translocation cavity

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Yang, M., Livnat Levanon, N., Acar, B. et al. Single-molecule probing of the conformational homogeneity of the ABC transporter BtuCD. Nat Chem Biol 14, 715–722 (2018). https://doi.org/10.1038/s41589-018-0088-2

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