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Published June 1, 2015 | Supplemental Material
Journal Article Open

A sulfur-based transport pathway in Cu^+-ATPases

Abstract

Cells regulate copper levels tightly to balance the biogenesis and integrity of copper centers in vital enzymes against toxic levels of copper. P_(IB)‐type Cu^+‐ATPases play a central role in copper homeostasis by catalyzing the selective translocation of Cu^+ across cellular membranes. Crystal structures of a copper‐free Cu^+‐ATPase are available, but the mechanism of Cu^+ recognition, binding, and translocation remains elusive. Through X‐ray absorption spectroscopy, ATPase activity assays, and charge transfer measurements on solid‐supported membranes using wild‐type and mutant forms of the Legionella pneumophila Cu^+‐ATPase (LpCopA), we identify a sulfur‐lined metal transport pathway. Structural analysis indicates that Cu^+ is bound at a high‐affinity transmembrane‐binding site in a trigonal‐planar coordination with the Cys residues of the conserved CPC motif of transmembrane segment 4 (C382 and C384) and the conserved Met residue of transmembrane segment 6 (M717 of the MXXXS motif). These residues are also essential for transport. Additionally, the studies indicate essential roles of other conserved intramembranous polar residues in facilitating copper binding to the high‐affinity site and subsequent release through the exit pathway.

Additional Information

© 2015 The Authors. Received 26 November 2014; Revised 13 March 2015; Accepted 31 March 2015. Published online 08.05.2015. D.M. was supported by an EMBO Short‐Term Fellowship (ASTF‐11‐2012) and the Graduate School of Science and Technology, Aarhus University. G.M. was supported by a Marie Curie International Outgoing Fellowship (European Commission, grant no. 252961). L.Z. was supported by an NSERC Postdoctoral Fellowship. Work at Caltech was supported by NIH grant GM45162 to D.C.R. We thank Prof. G.N. George (University of Saskatchewan, Canada), Prof. D. Winge (University of Utah, USA), and Prof. D.P. Giedroc (University of Indiana, USA) for providing the XANES spectra of model compounds, Cox17 and MtCsoR, respectively. We thank the staff at Beamline 7–3, Stanford Synchrotron Radiation Lightsource (SSRL). SSRL is operated for the DOE and supported by OBER and by the NIH, NIGMS (P41GM103393), and the NCRR (P41RR001209). M.R.M. and F.T.‐B. gratefully acknowledge financial support from the Ente Cassa di Risparmio di Firenze and the Italian Ministry of University and Research (PON01_00937). P.N. was supported by the BIOMEMOS advanced research grant of the European Research Council (250322). This project benefited from discussion with Nathan Dalleska and instrumentation made available by the Caltech Environmental Analysis Center. Author contributions DM and GM designed the experiments assisted by DCR, PG, and PN. Mutant constructs were generated by DM and LTP and DM and GM purified the protein samples. GM and LZ recorded the XAS data and analyzed the data together with DM; DM and GM performed and analyzed the stoichiometry experiments; DM and GM performed and analyzed the ATPase activity experiments. OS prepared membranes for charge transfer measurements. FT-B and MRM designed and performed concentration jump experiments on solidsupported membrane; FT-B analyzed and summarized charge transfer measurements. DM and GM summarized the results and prepared the manuscript figures, and DM, GM, and PN wrote the paper with comments from all authors. DCR, PG, and PN supervised the work.

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September 22, 2023
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