title: 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. PIB -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.
authors.affiliation: Centre for Membrane Pumps in Cells and Disease - PUMPkin, Department of Molecular Biology and Genetics, Danish National Research Foundation Aarhus University, Aarhus C, Denmark Division of Chemistry and Chemical Engineering and Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Mattle
authors.affiliation: Division of Chemistry and Chemical Engineering and Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA.
authors.forename: Limei
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Centre for Membrane Pumps in Cells and Disease - PUMPkin, Department of Molecular Biology and Genetics, Danish National Research Foundation Aarhus University, Aarhus C, Denmark.
authors.forename: Oleg
authors.initials: O
authors.lastname: Sitsel
authors.affiliation: Centre for Membrane Pumps in Cells and Disease - PUMPkin, Department of Molecular Biology and Genetics, Danish National Research Foundation Aarhus University, Aarhus C, Denmark.
authors.forename: Lotte Thue
authors.initials: LT
authors.lastname: Pedersen
authors.affiliation: Department of Chemistry 'Ugo Schiff', University of Florence, Sesto Fiorentino, Italy.
authors.forename: Maria Rosa
authors.initials: MR
authors.lastname: Moncelli
authors.affiliation: Department of Chemistry 'Ugo Schiff', University of Florence, Sesto Fiorentino, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Tadini-Buoninsegni
authors.affiliation: Centre for Membrane Pumps in Cells and Disease - PUMPkin, Department of Molecular Biology and Genetics, Danish National Research Foundation Aarhus University, Aarhus C, Denmark.
authors.forename: Pontus
authors.initials: P
authors.lastname: Gourdon
authors.affiliation: Division of Chemistry and Chemical Engineering and Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA.
authors.forename: Douglas C
authors.initials: DC
authors.lastname: Rees
authors.affiliation: Centre for Membrane Pumps in Cells and Disease - PUMPkin, Department of Molecular Biology and Genetics, Danish National Research Foundation Aarhus University, Aarhus C, Denmark.
authors.forename: Poul
authors.initials: P
authors.lastname: Nissen
authors.affiliation: Centre for Membrane Pumps in Cells and Disease - PUMPkin, Department of Molecular Biology and Genetics, Danish National Research Foundation Aarhus University, Aarhus C, Denmark Division of Chemistry and Chemical Engineering and Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA gmeloni@caltech.edu gmeloni@mbg.au.dk.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Meloni
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D001665:Binding Sites; D001692:Biological Transport; D027682:Cation Transport Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003300:Copper / Q000378:metabolism*; D016952:Legionella pneumophila / Q000201:enzymology* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013455:Sulfur / Q000378:metabolism*
chemical_list: D027682:Cation Transport Proteins; D013455:Sulfur; D003300:Copper; D000251:Adenosine Triphosphatases
keywords: Cu+ coordination; Cu+‐ATPase; P‐type ATPase; X‐ray absorption spectroscopy; charge transfer measurements; membrane protein transporter