Noooo00 II [10HHIc

0, copper binding domain I, transmembrane domain 0, functional domain

Fig. 1. Primary structure of ATP7B protein with key features. Human ATP7B consists of 1465 amino acids. The general features of P-type ATPases included in ATP7B are TGEA (phosphatase domain), DKTGT (phosphorylation domain), TGDN (ATP binding domain), and MVGDGVNDSP that connects the ATP-binding domain to the transmembrane segment. The unique features of ATP7B are six copper-binding sites, CPC and SEHPL involved with cation translocation, and eight transmembrane segments. The CPC domain locates within the sixth transmembrane region. Numbers in parentheses indicate amino acid positions of the functional domains.

P-Type ATPases translocate a variety of cations across cell membranes. This class of enzymes is characterized by formation of a covalent phosphorylated intermediate in their reaction cycle by the transfer of y-phosphate of ATP to an aspartic acid residue of the protein and is inhibited by vanadate (31). General features of P-type ATPases present in ATP7B are the TGEA motif (phosphatase domain), the DKTGT motif (phosphorylation domain), the TGDN motif (ATP-binding domain), and the sequence MXGDGXNDXP that connects the ATP-binding domain to the transmembrane segment. ATP7B is further classified as a heavy-metal-transporting P-type ATPase, which pumps copper, cadmium, or silver. The characteristic features of this class of ATPases are one to six repeated motifs for metal binding (GMTCXXC) at the N-terminus of the molecule, the CPC motif in the sixth transmembrane region, the SEHPL motif, and eight transmembrane segments (3,7). A single copy of the GMTCXXC motif is found in bacterial CopA, two in yeast Ccc2, three in nematode CUA-1, five in rat Atp7b, and six in ATP7A and ATP7B (3,6). The metal-binding motifs of ATP7B are specific for copper, and stoichiometric analysis showed that 6 mol of copper were bound to 1 mol of the N-terminus containing the six metal-binding regions of the protein (32). The metal-binding motifs in ATP7A and ATP7B are shown to play essential roles in copper transport by the complementation of yeast Ccc2 (11,15,33); however, each domain is not functionally equivalent. The CPC and SEHPL motifs of heavy-metal-transporting ATPases are assumed to be involved with metal translocation across membranes. This is supported by recent observations that mutations introduced into the CPC or SEHPL result in loss of ATP7B function in copper transport (12,15). This class of proteins is called P1-ATPases or CPx-type ATPases (3,7). The primary structure of ATP7B is shown in Fig. 1.

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