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Crystal structure of a copper-transporting PIB-type ATPase

Abstract

Heavy-metal homeostasis and detoxification is crucial for cell viability. P-type ATPases of the class IB (PIB) are essential in these processes, actively extruding heavy metals from the cytoplasm of cells. Here we present the structure of a PIB-ATPase, a Legionella pneumophila CopA Cu+-ATPase, in a copper-free form, as determined by X-ray crystallography at 3.2 Å resolution. The structure indicates a three-stage copper transport pathway involving several conserved residues. A PIB-specific transmembrane helix kinks at a double-glycine motif displaying an amphipathic helix that lines a putative copper entry point at the intracellular interface. Comparisons to Ca2+-ATPase suggest an ATPase-coupled copper release mechanism from the binding sites in the membrane via an extracellular exit site. The structure also provides a framework to analyse missense mutations in the human ATP7A and ATP7B proteins associated with Menkes’ and Wilson’s diseases.

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Figure 1: Overall structure of the L. pneumophila Cu + -ATPase LpCopA.
Figure 2: Details of CopA compared to the binding sites I and II in SERCA1a in the equivalent calcium-free state.
Figure 3: The cytoplasmic platform and the heavy-metal binding domain.
Figure 4: Proposed stages at the copper transport pathway of CopA.
Figure 5: Distribution of human ATP7A missense mutations associated with Menkes’ disease.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the L. pneumophila Lpg1024 CopA crystal structure have been deposited at the Protein Data Bank under accession code 3RFU.

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Acknowledgements

We thank A. Pauluhn, C. Schulze-Briese, T. Tomizaki and V. Olieric (SLS), T. Ursby, M. Thunnissen, J. Unge and D. Haase (MAXLAB), and U. Müller, M. Weiss and K. Paithankar (BESSY) for assistance with synchrotron data collection. Support was provided by the Danscatt program of the Danish Natural Science Research Council. We also thank C. Buchrieser for supplying the L. pneumophila Philadelphia genome; X. D. Su for discussions, A. M. Nielsen for technical assistance and J. L. Karlsen for support on crystallographic computing. We are thankful to T. Deva and K. Faxén for preliminary functional analysis of the protein. P.G. was supported by the Swedish Research Council, X.-Y.L. by the China Scholarship Council and J.P.M. and B.P.P. by the Carlsberg Foundation. P.N. was supported by an advanced research grant (Biomemos) of the European Research Council and at earlier stages by a Hallas-Møller stipend of the Novo Nordisk Foundation.

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P.G. initiated the project, designed the expression construct and developed the protein production protocol assisted by J.P.M. Protein purification, activity measurements, crystallization, data collection, structure determination, refinement, and overall analysis of results were designed and performed by P.G. and X.-Y.L. jointly. B.P.P. designed and performed the Molecular Replacement screening procedure to initiate phasing, and assisted in structure determination, refinement and structural analysis. T.S. and L.B.M. identified genetic data and collected phenotypic data from Menkes’ disease patients. P.N. designed and supervised the project, and analysed results. P.G., X.-Y.L., B.P.P. and P.N. wrote the paper and all authors commented on the paper.

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Correspondence to Lisbeth Birk Møller or Poul Nissen.

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Gourdon, P., Liu, XY., Skjørringe, T. et al. Crystal structure of a copper-transporting PIB-type ATPase. Nature 475, 59–64 (2011). https://doi.org/10.1038/nature10191

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