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Methionine motifs of copper transport proteins provide general and flexible thioether-only binding sites for Cu(I) and Ag(I)

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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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Acknowledgments

We thank the National Science Foundation (Grant CAREER 0449699) for funding these studies. K.J.F. also thanks the Sloan Foundation and the Camille and Henry Dreyfus Foundation. We thank Marina Dickens, Anthony Ribeiro, and Ronald Venters for assistance with NMR spectroscopy, and Terry Oas for many helpful discussions. Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource, a national user facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program. P.R.G. was supported by a grant from the Camille and Henry Dreyfus Foundation (Henry Dreyfus Teacher-Scholar Program) and by a grant from the National Institutes of Health to the state of South Carolina as part of NCRR’s INBRE program (P20 RR-016461).

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Authors and Affiliations

  1. Department of Chemistry, Duke University, P.O. Box 90346, Durham, NC, 27708, USA

    Jeffrey T. Rubino & Katherine J. Franz

  2. Department of Chemistry and Biochemistry, College of Charleston, 66 George St., Charleston, SC, 29424, USA

    Pamela Riggs-Gelasco

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  1. Jeffrey T. Rubino
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Correspondence to Katherine J. Franz.

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Rubino, J.T., Riggs-Gelasco, P. & Franz, K.J. Methionine motifs of copper transport proteins provide general and flexible thioether-only binding sites for Cu(I) and Ag(I). J Biol Inorg Chem 15, 1033–1049 (2010). https://doi.org/10.1007/s00775-010-0663-9

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