The Interactions Of Ag(l) With The Met-rich Motifs In The Extracellular Region Of HCtr1

Human copper transporter1（hCtr1） is a glycosylated membrane protein and amember of a highly conserved copper transporter family. The hCtr1is composed of190amino acids, including three putative transmembrane domains, an extracellularN-terminal region and an intracellular C-terminal region. A recent structural study bycryoelectron crystallography revealed that hCtr1forms a symmetrical homotrimerwith a channel-like architecture in order to allow import of Cu（I）. The extracellularN terminus of hCtr1contains two methionine （Met）-rich and two histidine （His）-richmotifs that are thought to be essential for the function of the transporter. Althoughconsiderable progress has been made toward understanding the structure-functionaspects of hCtr1-mediated Cu transport, details on the interaction of hCtr1proteinwith Ag at an atomic level is still poorly understood.In this work, we studied the coordination and thermodynamic characteristics ofAg（I） binding with a20-residue peptide including hCtr1-M2region and its mutantswith Met/Ala substitution in SDS micellar solution using CD, NMR and ITCmethods. The CD experiment shows that the WT peptide forms an-helix structurein SDS micelles. The2D-NMR experiments show that the peptide forms an-helixstructure in the C-terminal region and the N-terminal region is flexible in SDSmicelles. The paramagnetic probe experiment suggests that the residues from Gly1to Pro11are exposed to water and the residues from Met12to Asn20embedded inthe micelles. Both CD and NMR experiments indicate that the secondary structure ofWT changed upon adding the Ag（I）. In the NMR spectra of the peptide, we observethe downfield shifting of the peaks associated with the γ and ε protons of methionineresidues upon the addition of Ag（I）, suggesting that the thioethers of methioninesparticipate in coordination with Ag（I）. The residues of methionines at position7,8,9 and12of the N-terminal region provide a strong coordination in the binding withsilver, while the residue Met10is not involved in the coordination.We also perform the ITC experiments to further study of the interactionsbetween Ag（I） and the hCtr1-M2peptide. We obtain the thermodynamic parametersof the binding of WT and various mutant peptides with Ag（I） in SDS micellarsolution, such as the binding constant K, the number of binding sites n, enthalpychange H, entropy change S and Gibbs free energy change G. Through analysisof the thermodynamic data, we found that the WT peptide binds Ag（I） at1:1ratiowith a relative weak affinity (K=1.12×10⁵M^-1), driven by enthalpy change.Compared with the results of coordination of WT with Ag（I） in SDS micelles, thethermodynamic data of the mutants M7A, M8A and M12A are dramatically different（particularly in n and S） and those of M9A are largely different. The results of ITCfurther prove the results of the NMR, namely, the residue Met7, Met8and Met12（the methionine residues at the position40,41and45in hCtr1protein） play animportant role in the coordination of the hCtr1-M2peptide with silver, Met9（themethionine residue at the position42in hCtr1protein）also play a role in the bindingwith a relative weaker affinity, while Met10（Met43in hCtr1protein） is not involvedin the coordination.We hope that these findings will provide some insight into the mechanism ofhCtr1transporting Ag（I）.