Department of Biochemistry, Microbiology and Immunology, School of Medicine, Wayne State University Detroit, Michigan 48201, United States.
Biochemistry. 2020 Dec 1;59(47):4488-4498. doi: 10.1021/acs.biochem.0c00770. Epub 2020 Nov 15.
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.
ZntA 赋予细胞抵抗 Pb、Zn 和 Cd 等有毒浓度的能力。它是 P 型 ATP 酶转运蛋白超家族的成员，该超家族包括人类 Cu 转运蛋白 ATP7A 和 ATP7B。P 型 ATP 酶通常具有亲水的 N 端金属结合结构域和八个跨膜螺旋。曾报道过 ATP7B 的剪接变体，它在松果体中有 100 倍更高的夜间特异性表达；它缺失了整个 N 端结构域和前四个跨膜螺旋。在此，我们报告了我们在 ZntA 中创建的类似截断物 Δ231-ZntA 的发现。Δ231-ZntA 没有 和 活性，大大降低。它在跨膜位点每个二聚体结合一个金属离子，结合亲和力提高了 15-19000 倍，这表明 Δ231-ZntA 的二聚体结构相对于 ZntA 发生了显著变化。与 ZntA 相反，Cd 对 Δ231-ZntA 的亲和力最高。金属结合残基 Cys、Cys 和 Asp 的定点突变显示，金属结合位点具有相当大的灵活性，这三个残基中的任意两个都能够结合 Zn 和 Pb，而在 ZntA 中则不是。然而，Cd 仅结合 Cys 和 Asp，Cys 不参与 Cd 结合。三维同源模型显示 ZntA 和 Δ231-ZntA 二聚体结构之间存在显著差异，这有助于解释这些观察结果。因此，ZntA 和 P 型 ATP 酶中的前四个跨膜螺旋在维持正确的二聚体结构方面起着重要作用。
