The relative influence of protein unfolding on the Cu(i) affinity of trafficking and target sites for copper has been determined. For the copper metallochaperone Atx1 from Synechocystis PCC 6803 (a cyanobacterium), Saccharomyces cerevisiae and humans unfolding in urea results in a decrease in the Cu(i) affinity from (4-5) ? 10(17) M(-1) to (1-3) ? 10(16) M(-1) at pH 7. The affinities of the unfolded Atx1s are similar to those for CXXC-containing peptides. Partial unfolding, due to the loop 5 His61Lys mutation in Synechocystis Atx1, gives rise to a more limited decrease in Cu(i) affinity. For the copper target protein plastocyanin from Synechocystis, chemical unfolding results in the Cu(i) affinity decreasing by 5-orders of magnitude. This differential influence of protein unfolding on Cu(i) affinity is due to a more complex copper site structure in the target protein, including numerous interactions of non-coordinating residues with ligating amino acids. This second-coordination sphere is much simpler in the Atx1s with the main interaction provided by the loop 5 residue that tunes the Cu(i) affinity by altering the pK(a) of the C-terminal Cys ligand of the CXXC motif. This interaction and others are absent in the unfolded Atx1s and the two Cys ligands have pK(a) values reminiscent of free thiols (>8) resulting in lowered Cu(i) affinities at pH 7. Residues close to the active site of the thiol-disulfide oxidoreductase thioredoxin appear to lower the Cu(i) affinity of its CXXC motif to 3.1 ? 10(15) M(-1) at pH 7, presumably to prevent copper binding in vivo. The structure of a copper site, including the number and relative position of ligands in the primary structure and the complexity of the second-coordination sphere, results in dramatically different effects of unfolding on Cu(i) affinity that has important implications for copper homeostasis.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|