Identification of the transmembrane metal binding site in Cu+-transporting PIB-type ATPases

P-IB-type ATPases have an essential role maintaining copper homeostasis. Metal transport by these membrane proteins requires the presence of a transmembrane metal occlusion/binding site. Previous studies showed that Cys residues in the H6 transmembrane segment are required for metal transport. In this study, the participation in metal binding of conserved residues located in transmembrane segments H7 and H8 was tested using CopA, a model Cu+-ATPase from Archaeoglobus fulgidus. Four invariant amino acids in the central portion of H7 (Tyr(682) and Asn(683)) and H8 (Met(711) and Ser(715)) were identified as required for Cu+ binding. Replacement of these residues abolished enzyme activity. These proteins did not undergo Cu+-dependent phosphorylation by ATP but were phosphorylated by P-i in the absence of Cu+. Moreover, the presence of Cu+ could not prevent the enzyme phosphorylation by P-i. Other conserved residues in the H7-H8 region were not required for metal binding. Mutation of two invariant Pro residues had little effect on enzyme function. Replacement of residues located close to the cytoplasmic end of H7-H8 led to inactive enzymes. However, these were able to interact with Cu+ and undergo phosphorylation. This suggests that the integrity of this region is necessary for conformational transitions but not for ligand binding. These data support the presence of a unique transmembrane Cu+ binding/translocation site constituted by Tyr-Asn in H7, Met and Ser in H8, and two Cys in H6 of Cu+-ATPases. The likely Cu+ coordination during transport appears distinct from that observed in Cu+ chaperone proteins or catalytic/redox metal binding sites.