Micelle-bound structure of an extracellular Met-rich domain of hCtr1 and its binding with silver

Abstract

Silver is a toxic metal. It can accumulate in tissues as a contaminant in foods and air. The human copper transporter 1 (hCtr1), as an uptake protein of copper, plays an important role in the accumulation of silver in mammalian cells. The extracellular N-terminal domain of hCtr1 contains two methionine-rich motifs, in which the second methionine-rich motif (M2) has been demonstrated to be crucial for uptake of Ag(I) and Cu(I). In the present work, the structure of an M2-inclusive peptide from hCtr1 34–53 in SDS (sodium dodecyl sulfate) micellar solution and the interaction of the micelle-bound peptide with Ag(I) were studied using nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC) and circular dichroism (CD) methods. The details of the binding site, affinity, stoichiometry and thermodynamics of the interaction between the micelle-bound peptide and Ag(I) were explored using NMR and calorimetric measurements titrated by Ag(I), and the role of each methionine residue in the binding of M2 motif with Ag(I) was estimated by individual substitution of methionine by alanine. It was revealed that the hydrophobic C-terminal region of the peptide folds as an α-helix and is embedded in SDS micelles, while the N-terminal region containing the Met motif locates at the surface of the micelles and binds Ag(I) mostly by Met40, Met41, Met43 and Met45. Differences between the peptide in the micelle-bound state and the peptide in the solution state with regards to the binding affinity, stoichiometry and thermodynamics were observed. It is suggested that the interaction between the extracellular domain of hCtr1 and membrane surface may play an important role for the binding structure and binding affinity of the M2 motif with Ag(I).