Ion Selectivity by Heavy Metal Transport ATPases

描述(由申请人提供)：本项目的目标是 确定决定离子选择性和调节的结构特征 重金属(过渡)转运ATPase的机制(CPX-ATPase)。这些 P型ATPase转运金属(铜、银、锌、二氧化碳、镉、铅) 生物膜和它们的浓度梯度。这些是关键的酶 在重金属的吸收、分布和生物积累机制中。 该蛋白家族的成员对威尔逊和门克斯病负有责任 在人类身上。在原核生物中发现了更简单的CPX-ATPase，它们看起来像 适合这一系列运输机的型号。一种跨膜金属结合 “cpx”位点([C/S]P[C/H/S])和1-6个细胞质金属结合重复序列 该蛋白的N-末端是CPX-ATPase的特征。虽然推定 根据CPX-ATPase的结构，提出了其转运机制 与其他P型ATPase(Na，K-ATPase，Ca-ATPase)的同源性及其转运 金属离子专一性的化学计量学、调节机制和决定因素 都是未知的。在这个提议中，使用原核生物酶和不同的 预测离子选择性，我们计划测试特征CPX序列是否 和/或第七和第八跨膜片段中的保守序列 蛋白质决定离子的专一性。此外，我们将测试推定的 胞质金属结合部位在离子选择性中的作用 监管领域。这些研究的结果将有助于建立 重金属与载体蛋白结合的机制和增加我们的 对重金属迁移的理解。

DESCRIPTION (provided by applicant): The objective of this project is to identify structural features that determine ion selectivity and regulation mechanisms of heavy (transition) metal transport ATPases (CPx-ATPases). These P-type ATPases transport metals (Cu+, Ag+, Zn2+, Co2+, Cd2+, Pb2+) across biomembranes and against their concentration gradients. These are key enzymes in the mechanisms of heavy metal absorption, distribution, and bioaccumulation. Members of this protein family are responsible for Wilson and Menkes' disease in humans. Simpler CPx-ATPases are found in prokaryotes and they appear as suitable models for this family of transporters. A transmembrane metal binding "CPx" site ([C/S]P[C/H/S]) and one to six cytoplasmic metal binding repeats in the N-terminus of the protein characterize the CPx-ATPases. While putative transport mechanism has been proposed for CPx-ATPases based on their structural homology with other P-type ATPases (Na,K-ATPase, Ca-ATPase), their transport stoichiometry, regulatory mechanisms, and determinants of metal ion specificity are not known. In this proposal, using prokaryote enzymes with different predicted ion selectivity, we plan to test whether the signature CPx sequence and/or conserved sequences in the seventh and eighth transmembrane segments of the protein determine ion specificity. In addition, we will test the putative role of the cytoplasmic metal binding site in ion selectivity and as a regulatory domain. Results from these studies will help to establish the mechanism of heavy metal binding to carrier proteins and increase our understanding of heavy metal transport.