Structures Of Copper Transporters And Their Roles In Silver Transmission

Copper is an essential trace metal element for human beings. Human copper transporter 1(hCtr1) plays an important role in high-affinity uptake of cellular copper. Besides cuprous, Ctr1 can also transport the silver ion. Ctr1 is characterized as a homomultimer that forms a cone-shaped pore in the plasma membrane with a narrow extracellular entrance and a wider export on the cytosolic side. The protein is separated to an extracellular N-terminal domain, three transmembrane domains, and an intracellular carboxyl-terminal domain. The N-terminal domain includes multiple methionines and histidines that are significant for the recruitment of copper to the entry of the pore. The TMD2 with a 150MXXX154 M motif lines the pore of the trimer and has been proved to be crucial for the function of the protein. Human copper transporter 2(hCtr2) is a low affinity transporter with a majority located in late endosome and lysosome compartments and a small proportion distributed at the plasma membrane. Although hCtr2 shares only 41% amino acid homology with hCtr1 and lacks the N- and C-terminal domain characteristic of Ctr1, the residues in transmembrane domains that are known to be essential for the transport function of Ctr1 are conserved in Ctr2. Therefore, hCtr2 is believed to have a topological and structural homology to hCtr1, characterized by a homotrimer with TMD2 lining the channel-like pore. However, no evidences are currently available.To understand the significance of TMD2 in the pore construction and transport function of hCtr1/hCtr2 and the roles of the N-terminal domain of the proteins in the trafficking of Ag(I)/Cu(I), we studied the structure and assembly of hCtr1-TMD2, hCtr2-TMD2 and hCtr2-N-domain in SDS micelles and the chemical stoichiometry, affinity and thermodynamic parameters of the binding of the micelle-bound peptide to Ag(I) using NMR, ITC, CD and SDS-PAGE techniques. The results obtained in these studies are summarized as follows:1. Isolated hCtr1-TMD2 peptide can form a trimer in SDS micelles and each peptide in trimer is ?-helical. The trimer binds Ag(I) with a chemical stoichiometry of 3:2. Such a binding is formed possibly by two triad coordinations of the Met residues in the MXXXM motif of the C-terminal part of the TMD2 peptide. The structure of the peptide is slightly changed after binding with Ag(I). The conversion from methionine to leucine in the MXXXM motif either at one site or at two sites results in a decrease in the aggregation propensity and the binding affinity of the peptide, suggesting that the two methionine residues in the 150MXXX154 M motif of hCtr1 may be important for TMD2 both in the trimeric assembly and in a higher-affinity binding to Ag(I).2. The TMD2 peptide from hCtr2 also forms an ?-helical structure in SDS micelles. The aggregation state of the peptide depends on the peptide/SDS ratio. Dimer is a predominant oligomeric state at a peptide/SDS of 1:120, while only trimer is observed when the peptide/SDS ratio is increased to 1:20. Both peptide dimer and trimer can bind Ag(I) with a chemical stoichiometry of 1:1 and 3:2, respectively, and the binding affinity of trimer is larger than that of dimer. Two Met residues in the MXXXM motif of hCtr2-TMD2 are involved in the coordination with Ag(I) directly. Importantly, hCtr2-TMD2 displays a waker aggregation propensity and a lower binding affinity for Ag(I) than hCtr1-TMD2 at the same peptide/SDS.3. The extracellular N-terminal domain of hCtr2 forms a β-sheet predominated structure in SDS micelles and can bind with silver ion to a 1:1 binding stoichiometry. The binding has little effect on the secondary structure of the peptide. In contrast, the peptide forms a random coil in bulk aqueous solution. The binding with Ag(I) leads to a decrease in the random coil content and an increase in β-sheet component. Two Met residues in the N-terminal end(1MetX3Met) are critical for the binding of the peptide with Ag(I). The change in pH values exerts no influence on the second structure of the peptide, but has an effect on the binding ability of the peptide with the silver ion.The finding in our work may be significant for further understanding of the structure-function relationship and the mechanism underlying the cellular uptake of Cu(I)/Ag(I) by copper transporters.