Structural Role of the First Four Transmembrane Helices in ZntA, a P-Type ATPase from .

ZntA from confers resistance to toxic concentrations of Pb, Zn, and Cd. It is a member of the P-ATPase transporter superfamily, which includes the human Cu-transporting proteins ATP7A and ATP7B. P-type ATPases typically have a hydrophilic N-terminal metal-binding domain and eight transmembrane helices. A splice variant of ATP7B was reported, which has 100-fold higher night-specific expression in the pineal gland; it lacks the entire N-terminal domain and the first four transmembrane helices. Here, we report our findings with Δ231-ZntA, a similar truncation we created in ZntA. Δ231-ZntA has no and greatly reduced activity. It binds one metal ion per dimer at the transmembrane site, with a 15-19000-fold higher binding affinity, indicating highly significant changes in the dimer structure of Δ231-ZntA relative to that of ZntA. Cd has the highest affinity for Δ231-ZntA, in contrast to ZntA, which has the highest affinity for Pb. Site-specific mutagenesis of the metal-binding residues, Cys, Cys, and Asp, showed that there is considerable flexibility at the metal-binding site, with any two of these three residues able to bind Zn and Pb unlike in ZntA. However, Cd binds to only Cys and Asp, with Cys not involved in Cd binding. Three-dimensional homology models show that there is a dramatic difference between the ZntA and Δ231-ZntA dimer structures, which help to explain these observations. Therefore, the first four transmembrane helices in ZntA and P-type ATPases play an important role in maintaining the correct dimer structure.