The metal-binding sites of the zinc-transporting P-type ATPase of Escherichia coli. Lys 693 and Asp 714 in the seventh and eighth transmembrane segments of ZntA contribute to the coupling of metal binding and ATPase activity

Abstract

ZntA is a P-type ATPase which transports Zn 2+, Pb 2+ and Cd 2+ out of the cell. Two cysteine-containing motifs, CAAC near the N-terminus and CPC in transmembrane helix 6, are involved in binding of the translocated metal. We have studied these motifs by mutating the cysteines to serines. The roles of two other possible metal-binding residues, K 693 and D 714, in transmembrane helices 7 and 8, were also addressed. The mutation CAAC → SAAS reduces the ATPase activity by 50%. The SAAS mutant is phosphorylated with ATP almost as efficiently as the wild type. However, its phosphorylation with P i is poorer than that of the wild type and its dephosphorylation rate is faster than that of the wild type ATPase. The CPC → SPS mutant is inactive but residual phosphorylation with ATP could still be observed. The most important findings of this work deal with the prospective metal-binding residues K 693 and D 714: the substitution K693N eliminates the Zn 2+-stimulated ATPase activity completely, although significant Zn 2+-dependent phosphorylation by ATP remains. The K693N ATPase is hyperphosphorylated by P i. ZntA carrying the change D714M has strong metal-independent ATPase activity and is very weakly phosphorylated both by ATP and P i. In conclusion, K 693 and D 714 are functionally essential and appear to contribute to the metal specificity of ZntA, most probably by being parts of the metal-binding site made up by the CPC motif.