Regensburg 2016 – wissenschaftliches Programm
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BP: Fachverband Biologische Physik
BP 3: Protein Structure and Dynamics
BP 3.4: Vortrag
Montag, 7. März 2016, 10:30–10:45, H44
The C-terminus of human copper importer, Ctr1, acts as binding site and transfers copper to Atox1 — •Michael Kovermann1,2, Dana Kahra2, and Pernilla Wittung-Stafshede2,3 — 1Fachbereich Chemie, Universität Konstanz, Germany — 2Department of Chemistry, Umeå University, Sweden — 3Department of Biology and Bioengineering, Chalmers University of Technology, Sweden
Uptake of copper ions (Cu) into human cells is mediated by the plasma membrane protein Ctr1, 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 a HCH motif thought to interact with Cu. We here employ biophysical experiments under anaerobic conditions to peptide models of the Ctr1 C-terminus to deduce Cu-binding residues, Cu affinity and 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 an one-to-one 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 does 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.