title: The C-Terminus of Human Copper Importer Ctr1 Acts as a Binding Site and Transfers Copper to Atox1.
abstract: Uptake of copper (Cu) ions into human cells is mediated by the plasma membrane protein Ctr1 and is followed by Cu transfer to cytoplasmic Cu chaperones for delivery to Cu-dependent enzymes. The C-terminal cytoplasmic tail of Ctr1 is a 13-residue peptide harboring an HCH motif that is thought to interact with Cu. We here employ biophysical experiments under anaerobic conditions in peptide models of the Ctr1 C-terminus to deduce Cu-binding residues, Cu affinity, and the ability to release Cu to the cytoplasmic Cu chaperone Atox1. Based on NMR assignments and bicinchoninic acid competition experiments, we demonstrate that Cu interacts in a 1:1 stoichiometry with the HCH motif with an affinity, KD, of ∼10(-14) M. Removing either the Cys residue or the two His residues lowers the Cu-peptide affinity, but site specificity is retained. The C-terminal peptide and Atox1 do not interact in solution in the absence of Cu. However, as directly demonstrated at the residue level via NMR spectroscopy, Atox1 readily acquires Cu from the Cu-loaded peptide. We propose that Cu binding to the Ctr1 C-terminal tail regulates Cu transport into the cytoplasm such that the metal ion is only released to high-affinity Cu chaperones.
authors.affiliation: Chemistry Department, Umeå University, Umeå, Sweden.
authors.forename: Dana
authors.initials: D
authors.lastname: Kahra
authors.affiliation: Chemistry Department, Umeå University, Umeå, Sweden; Chemistry Department, University of Konstanz, Konstanz, Germany. Electronic address: michael.kovermann@uni-konstanz.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Kovermann
authors.affiliation: Chemistry Department, Umeå University, Umeå, Sweden; Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden. Electronic address: pernilla.wittung@chalmers.se.
authors.forename: Pernilla
authors.initials: P
authors.lastname: Wittung-Stafshede
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001692:Biological Transport; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D000079922:Copper Transport Proteins; D000079923:Copper Transporter 1; D006801:Humans; D056511:Metallochaperones / Q000378:metabolism*; D018832:Molecular Chaperones; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding
chemical_list: C474500:ATOX1 protein, human; D027682:Cation Transport Proteins; D000079922:Copper Transport Proteins; D000079923:Copper Transporter 1; D056511:Metallochaperones; D018832:Molecular Chaperones; D010446:Peptide Fragments; C490079:SLC31A1 protein, human; D003300:Copper