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Crystal structure of a heterodimeric ABC transporter in its inward-facing conformation

Nature Structural & Molecular Biology volume 19, pages 395–402 (2012)Cite this article

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Abstract

ATP-binding cassette (ABC) transporters shuttle a wide variety of molecules across cell membranes by alternating between inward- and outward-facing conformations, harnessing the energy of ATP binding and hydrolysis at their nucleotide binding domains (NBDs). Here we present the 2.9-Å crystal structure of the heterodimeric ABC transporter TM287–TM288 (TM287/288) from Thermotoga maritima in its inward-facing state. In contrast to previous studies, we found that the NBDs only partially separate, remaining in contact through an interface involving conserved motifs that connect the two ATP hydrolysis sites. We observed AMP-PNP binding to the degenerate catalytic site, which deviates from the consensus sequence in the same positions as the eukaryotic homologs CFTR and TAP1–TAP2 (TAP1/2). The TM287/288 structure provides unprecedented insights into the mechanism of heterodimeric ABC exporters and will enable future studies on this large transporter superfamily.

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Figure 1: Functional analysis of TM287/288.
Figure 2: TM287/288 viewed along the membrane plane.
Figure 3: Arrangement of the NBDs.
Figure 4: Preferential nucleotide binding to the degenerate site of TM287/288.
Figure 5: Conformational changes from the inward-facing state of TM287/288 to the outward-facing state (Sav1866).
Figure 6: Proposed mechanism of transport in heterodimeric ABC transporters.

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Acknowledgements

We thank A. Mittal, C. Madhurantakam and E. Geertsma for helpful discussions and R. Dutzler for critical reading of the manuscript. We acknowledge B. Blattmann and C. Stutz-Ducommun from the NCCR crystallization facility for crystal screening and the staff of the X06SA beamline at the Swiss Light Source of the Paul Scherrer Institute for support during data collection. We thank H. van Veen (Department of Pharmacology, University of Cambridge) for the L. lactis NZ9000 ΔlmrA ΔlmrCD strain. This work was funded by the Swiss NCCR Structural Biology program, an Ambizione grant of the Swiss National Science Foundation (to M.A.S.) and a Forschungskredit of the University of Zürich (to M.A.S.). M.H. is affiliated with the PhD program in Biomolecular Structure and Mechanism of the Life Science Zürich Graduate School.

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  1. Department of Biochemistry, University of Zürich, Zürich, Switzerland.,

    Michael Hohl, Christophe Briand, Markus G Grütter & Markus A Seeger

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Contributions

M.H., M.G.G. and M.A.S. conceived the project. M.H. and M.A.S. cloned, purified and crystallized TM287/288 and carried out the functional assays. M.H., C.B. and M.A.S. collected and processed the crystallographic data. C.B., M.G.G. and M.A.S. solved the structure and built the model. All authors interpreted the structure and wrote the manuscript.

Corresponding authors

Correspondence to Markus G Grütter or Markus A Seeger.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Methods (PDF 9323 kb)

Supplementary Video 1

Morph between the structure of the NBDs of TM287/288 (inward-facing conformation) and a homology model based on the Sav1866 structure (outward-facing conformation) as seen from the top. Catalytic residues of the consensus site and AMP-PNP are depicted as sticks. (AVI 9723 kb)

Supplementary Video 2

Morph between the structure of TM287/288 (inward-facing conformation) and a homology model based on the Sav1866 structure (outward-facing conformation) from two sides and from the top. AMP-PNP is shown as sticks. (AVI 20180 kb)

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Hohl, M., Briand, C., Grütter, M. et al. Crystal structure of a heterodimeric ABC transporter in its inward-facing conformation. Nat Struct Mol Biol 19, 395–402 (2012). https://doi.org/10.1038/nsmb.2267

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