Reconstruction of a helical trimer by the second transmembrane domain of human copper transporter 2 in micelles and the binding of the trimer to silver

Copper transporter 2 (Ctr2) is identified as a homologue of copper transporter 1 (Ctr1) and believed to transport copper and silver ions by a similar mechanism to Ctr1. Of three pore-forming transmembrane domains, the second transmembrane domain (TMD2) is highly conserved by the two proteins and the MXXXM motif in TMD2 is significant for the transport function of the proteins. However, whereas Ctr1 is a high-affinity transporter, Ctr2 is a low-affinity transporter. Herein, we studied the structure and assembly of the peptide corresponding to TMD2 of human Ctr2 (hCtr2) in sodium dodecyl sulfate (SDS) micelles and the binding of the micelle-bound peptide to silver ion using nuclear magnetic resonance, circular dichroism, isothermal titration calorimetry and electrophoresis. The peptide hCtr2-TMD2 formed an a-helix in SDS micelles, and could assemble mainly as a dimer at a lower molar ratio of peptide : SDS or a trimer at a higher peptide : SDS ratio. The peptide trimer could bind two silver ions by the coordination of the C-terminal part and two methionine residues in the MXXXM motif played an important role in the binding. Our results showed that the hCtr2-TMD2 trimer has weaker intermolecular interactions and a lower binding affinity for silver ion compared with the results of our previous study on hCtr1-TMD2. This finding suggests that the intermolecular interactions between the second transmembrane domains may play a significant role in the pore formation of the two transporters and the binding affinity for Ag(I).