Structural Role of the First Four Transmembrane Helices in ZntA, a P_1B-Type ATPase from Escherichia coli.

ZntA from Escherichia coli confers resistance to toxic concentrations of Pb²⁺, Zn²⁺, and Cd²⁺. It is a member of the P_1B-ATPase transporter superfamily, which includes the human Cu⁺-transporting proteins ATP7A and ATP7B. P_1B-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 in vivo and greatly reduced in vitro 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. Cd²⁺ has the highest affinity for Δ231-ZntA, in contrast to ZntA, which has the highest affinity for Pb²⁺. Site-specific mutagenesis of the metal-binding residues, ³⁹²Cys, ³⁹⁴Cys, and ⁷¹⁴Asp, showed that there is considerable flexibility at the metal-binding site, with any two of these three residues able to bind Zn²⁺ and Pb²⁺ unlike in ZntA. However, Cd²⁺ binds to only ³⁹²Cys and ⁷¹⁴Asp, with ³⁹⁴Cys not involved in Cd²⁺ 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_1B-type ATPases play an important role in maintaining the correct dimer structure.

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