Methionine motifs of copper transport proteins provide general and flexible thioether-only binding sites for Cu(I) and Ag(I) |
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Authors: | Jeffrey T Rubino Pamela Riggs-Gelasco Katherine J Franz |
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Institution: | (1) Department of Chemistry, Duke University, P.O. Box 90346, Durham, NC 27708, USA;(2) Department of Chemistry and Biochemistry, College of Charleston, 66 George St., Charleston, SC 29424, USA; |
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Abstract: | Cellular acquisition of copper in eukaryotic organisms is primarily accomplished through high-affinity copper transport proteins
(Ctr). The extracellular N-terminal regions of both human and yeast Ctr1 contain multiple methionine residues organized in
copper-binding Mets motifs. These motifs comprise combinations of methionine residues arranged in clusters of MXM and MXXM,
where X can be one of several amino acids. Model peptides corresponding to 15 different Mets motifs were synthesized and determined
to selectively bind Cu(I) and Ag(I), with no discernible affinity for divalent metal ions. These are rare examples of biological
thioether-only metal binding sites. Effective dissociation constant (K
D) values for the model Mets peptides and Cu(I) were determined by an ascorbic acid oxidation assay and validated through electrospray
ionization mass spectrometry and range between 2 and 11 μM. Affinity appears to be independent of pH, the arrangement of the
motif, and the composition of intervening amino acids, all of which reveal the generality and flexibility of the MX1–2MX1–2M domain. Circular dichroism spectroscopy, 1H-NMR spectroscopy, and X-ray absorption spectroscopy were also used to characterize the binding event. These results are
intended to aid the development of the still unknown mechanism of copper transport across the cell membrane. |
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