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（过渡）金属转运ATP酶（CPx-ATP酶）。这些 P型ATP酶跨膜转运金属（Cu+、Ag+、Zn 2+、Co 2+、Cd 2+、Pb 2+） 生物膜和浓度梯度。这些是关键酶 重金属的吸收、分布和生物富集机制。 该蛋白质家族的成员是导致威尔逊和门克斯病的原因 在人类身上。在原核生物中发现了更简单的CPx-ATP酶， 适合这类运输机的型号。跨膜金属结合 “CPx”位点（[C/S]P[C/H/S]）和1至6个细胞质金属结合重复序列， 蛋白质的N-末端表征CPx-ATP酶。虽然假定 CPx-ATP酶的运输机制已根据其结构提出 与其它P型ATP酶（Na，K-ATP酶，Ca-ATP酶）同源，它们的转运 化学计量、调节机制和金属离子特异性的决定因素 不知道。在这个提议中，使用具有不同的 预测的离子选择性，我们计划测试是否签名CPx序列 和/或第七和第八跨膜区段中的保守序列， 蛋白质决定离子特异性。此外，我们将测试假定的 细胞质金属结合位点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，并增加我们的 了解重金属运输。

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.