Human copper transporter 1 (hCtr1) transports copper and silver by a homotrimer. The protein contains three transmembrane domains in which the second transmembrane domain (TMD2) is a key component lining the central pore of the trimer. The MXXXM motif in the C-terminal end of TMD2 plays a significant role in the function of hCtr1. In this study, we characterized the structure and assembly of isolated TMD2 of hCtr1 in sodium dodecyl sulfate (SDS) micelles and the interaction of the micelle-bound peptide with silver ion using nuclear magnetic resonance, circular dichroism, isothermal titration calorimetry and electrophoresis techniques. We detected the formation of a trirner of the isolated hCtr1-TMD2 in SDS micelles and the binding of the trimer to Ag(I) by a chemical stoichiometry of 3:2 of peptide:Ag(I). We showed that either an intensive pretreatment of the TMD2 peptide by 1,1,1,3,3,3-hexafluoro-2-propanol solvent or a conversion from methionine to leucine in the MXXXM motif changes the aggregation structure of the peptide and decreases the binding affinity by 1 order of magnitude. Our results suggest that the intrinsic interaction of the second transmembrane domain itself may be closely associated with the formation of hCtr1 pore in cellular membranes, and two methionine residues in the MXXXM motif may be important for TMD2 both in the trimeric assembly and in a higher-affinity binding to Ag(I).