Article

Conformational dynamics in substrate-binding domains influences transport in the ABC importer GlnPQ

  • Nature Structural & Molecular Biology volume 22, pages 5764 (2015)
  • doi:10.1038/nsmb.2929
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Abstract

The conformational dynamics in ABC transporters is largely elusive. The ABC importer GlnPQ from Lactococcus lactis has different covalently linked substrate-binding domains (SBDs), thus making it an excellent model system to elucidate the dynamics and role of the SBDs in transport. We demonstrate by single-molecule spectroscopy that the two SBDs intrinsically transit from open to closed ligand-free conformation, and the proteins capture their amino acid ligands via an induced-fit mechanism. High-affinity ligands elicit transitions without changing the closed-state lifetime, whereas low-affinity ligands dramatically shorten it. We show that SBDs in the closed state compete for docking onto the translocator, but remarkably the effect is strongest without ligand. We find that the rate-determining steps depend on the SBD and the amino acid transported. We conclude that the lifetime of the closed conformation controls both SBD docking to the translocator and substrate release.

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Acknowledgements

We thank S. Weiss (University of California Los Angeles) and A.N. Kapanidis (University of Oxford) for providing software for data acquisition and analysis of ALEX data. This work was supported by grants from the Netherlands Organization for Scientific research (NWO; Top-subsidy grant 700.56.302 to B.P.), the Marie-Curie Initial Training Networks program Network for Integrated Cellular Homeostasis (NICHE) (coordinated by B.P.) and the Zernike Institute for Advanced Materials and the Centre for Synthetic Biology (University of Groningen startup grant to T.C.). G.G. is supported by a NWO-VENI grant (grant no. 722.012.012). E.P. acknowledges a postdoctoral fellowship from the German Science Foundation (DFG; grant PL696/2-1). We thank R.M. Scheek for assistance with the data analysis.

Author information

Author notes

    • Giorgos Gouridis
    • , Gea K Schuurman-Wolters
    •  & Evelyn Ploetz

    These authors contributed equally to this work.

Affiliations

  1. Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, the Netherlands.

    • Giorgos Gouridis
    • , Gea K Schuurman-Wolters
    • , Ruslan Vietrov
    • , Marijn de Boer
    •  & Bert Poolman
  2. Molecular Microscopy Research Group, Zernike Institute for Advanced Materials, University of Groningen, Groningen, the Netherlands.

    • Giorgos Gouridis
    • , Evelyn Ploetz
    • , Florence Husada
    • , Marijn de Boer
    •  & Thorben Cordes

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Contributions

G.G., G.K.S.-W., E.P., M.d.B., T.C. and B.P. designed experiments and analyzed and interpreted the data. G.G., G.K.S.-W., E.P., F.H., R.V. and T.C. performed experiments. G.G., G.K.S.-W., E.P., T.C. and B.P. wrote the manuscript. All authors approved the final version of the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Thorben Cordes or Bert Poolman.

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–6, Supplementary Tables 1 and 2, and Supplementary Notes 1 and 2

  2. 2.

    Supplementary Data Set 1

    Full western blots used in the main text