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Human copper transporter 1 (hCtr1)transports copper and silverby a homotrimer. The protein contains three transmembrane domainsin which the second transmembrane domain (TMD2) is a key componentlining the central pore of the trimer. The MXXXM motif in the C-terminalend of TMD2 plays a significant role in the function of hCtr1. Inthis study, we characterized the structure and assembly of isolatedTMD2 of hCtr1 in sodium dodecyl sulfate (SDS) micelles and the interactionof the micelle-bound peptide with silver ion using nuclear magneticresonance, circular dichroism, isothermal titration calorimetry andelectrophoresis techniques. We detected the formation of a trimerof the isolated hCtr1-TMD2 in SDS micelles and the binding of thetrimer to Ag(I) by a chemical stoichiometry of 3:2 of peptide:Ag(I).We showed that either an intensive pretreatment of the TMD2 peptideby 1,1,1,3,3,3-hexafluoro-2-propanol solvent or a conversion frommethionine to leucine in the MXXXM motif changes the aggregation structureof the peptide and decreases the binding affinity by 1 order of magnitude.Our results suggest that the intrinsic interaction of the second transmembranedomain itself may be closely associated with the formation of hCtr1pore in cellular membranes, and two methionine residues in the MXXXMmotif may be important for TMD2 both in the trimeric assembly andin a higher-affinity binding to Ag(I). [ABSTRACT FROM AUTHOR]