Abstract
ATP7B, the Wilson disease-associated Cu(I)-transporter, and ZntA from Escherichia coli are soft metal P1-type ATPases with mutually exclusive metal ion substrates. P1-type ATPases have a distinctive amino-terminal domain containing the conserved metal-binding motif GXXCXXC. ZntA has one copy of this motif while ATP7B has six copies. The effect of interchanging the amino-terminal domains of ATP7B and ZntA was investigated. Chimeric proteins were constructed in which either the entire amino-terminal domain of ATP7B or only its sixth metal-binding motif replaced the amino-terminal domain of ZntA. Both chimeras conferred resistance to lead, zinc, and cadmium salts but not to copper salts. The purified chimeras displayed activity with lead, cadmium, zinc, and mercury, which are substrates of ZntA. There was no activity with copper or silver, which are substrates of ATP7B. The chimeras were 2-3-fold less active than ZntA. Thus, the amino-terminal domain of P1-type ATPases cannot alter the metal specificity determined by the transmembrane segment. Also, these results suggest that this domain interacts with the rest of the transporter in a metal ion-specific manner; the amino-terminal domain of ATP7B cannot replace that of ZntA in restoring full catalytic activity.
Highlights
P1-type ATPases, a subgroup of P-type ATPases, transport soft metal ions such as copper, silver, zinc, cadmium, lead, and cobalt across biological membranes (1– 4)
The amino-terminal domain of P1-type ATPases cannot alter the metal specificity determined by the transmembrane segment. These results suggest that this domain interacts with the rest of the transporter in a metal ion-specific manner; the amino-terminal domain of ATP7B cannot replace that of ZntA in restoring full catalytic activity
The Vmax values obtained for both chimeras are lower than the values obtained for ZntA, especially in the presence of thiolates in the assay buffer. These results confirm our previous conclusion that metal ion specificity is determined by the transmembrane part of the ATPase; the amino-terminal domain cannot override this intrinsic specificity. These results demonstrate that the aminoterminal domain interacts with the rest of the transporter in a metal ion-specific manner; the amino-terminal domain of ATP7B cannot replace that of ZntA in restoring full catalytic activity
Summary
P1-type ATPases, a subgroup of P-type ATPases, transport soft metal ions such as copper, silver, zinc, cadmium, lead, and cobalt across biological membranes (1– 4). Our goal was to investigate whether the amino-terminal domain of P1-type ATPases determines specificity toward particular metal ions Toward this end, we constructed chimeric proteins in which the amino-terminal domain of ZntA was replaced by the amino-terminal domain of the Wilson Cu(I)-transporter ATP7B (Fig. 1). It has been reported that the sixth metal-binding domain is necessary and sufficient for the transport activity of ATP7B (22) Both chimeric proteins were able to confer resistance toward Pb(II), Cd(II), and Zn(II) in a zntA-disrupted E. coli strain but not to copper in an E. coli strain disrupted in copA, which encodes a Cu(I)-transporting ATPase (23). These results demonstrate that the aminoterminal domain interacts with the rest of the transporter in a metal ion-specific manner; the amino-terminal domain of ATP7B cannot replace that of ZntA in restoring full catalytic activity
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