Mechanistic steps of metal uploading into Cu+‐transporting ATPases

Abstract

Mutations in the two human Cu+‐ATPases lead to either Wilson's or Menkes' diseases. Cu+‐ATPases accept cytoplasmic Cu+ from Cu+‐chaperones and subsequently drive this metal transmembrane transport. It has been postulated that cytosolic metal binding domains (MBDs) have the regulatory role based on their Cu+‐dependent interaction with the ATP‐binding domain. Alternatively, they might affect chaperone‐ATPase interaction. Here, we show that MBDs do not play a role in Cu+ delivery nor affect the interaction of the chaperone with the ATPase. Moreover, nucleotides, as Cu+, prevent the MBDs interaction with the ATP‐binding domain. Interestingly, we also observed that for the chaperone to upload Cu+ to the two transmembrane metal binding sites (TM‐MBSs) present in Cu+‐ATPases, the presence of nucleotides is required. Our results suggest that Cu+ loading of the TM‐MBS occurs in a sequential manner, in which after metal binding to one of the two sites, ATP binding is necessary to make the second site accessible for chaperone‐bound Cu+. This is in sharp contrast with "classical" P‐type ATPases, such as the Ca+2‐ATPase, in which ion uploading is independent of ATP binding. This work was supported by NSF grant MCB‐0743901.