STRUCTURE OF THE NUCLEOTIDE-BINDING DOMAINS OF HUMAN COPPER-TRANSPORTING ATPASES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 本课题的目的是研究肝豆状核变性相关蛋白和门克斯病相关蛋白这两种人类铜转运ATP酶的核苷酸结合域的结构，并阐明ATP结合引起的结构域的构象变化。铜转运ATP酶属于一类独特且研究较少的膜转运酶（Lutsenko等人，2002），并且是人体组织中铜代谢所必需的。编码这些蛋白质的ATP 7A和ATP 7 B基因的突变导致严重的遗传性代谢紊乱。目前还没有高分辨率的重金属转运ATP酶的结构，核苷酸结合结构域的结构将在无核苷酸和ATP结合形式下得到解决。 将确定ATP结合位点在结构域的三维结构中的位置。 将在P型ATP酶的催化循环的背景下研究ATP结合后发生的构象变化，重点是将重金属转运ATP酶与研究良好的Ca 2 +-ATP酶分离的独特功能。将确定与一级结构的非同源区域相关的Wilson病和Menkes病蛋白的核苷酸结合结构域的三维结构的差异。该项目还将为未来研究铜转运ATP酶中致病突变的结构效应，解决Wilson和Menkes病蛋白质较大结构域的结构和绘制结构域相互作用表面提供基础。 迄今为止，包含残基1036-1196的威尔逊病ATP酶（WNABD）的N-核苷酸结合结构域的结构已经以ATP结合形式被解析（Dmitriev等人，2006年）。用于确定N结构域的两种致病突变变体（E1064 A和H1069 A）的结构的数据收集和处理正在进行中参考文献 1. Dmitriev，O.，齐夫科夫斯基河，Abildgaard，F.，摩根角T.，Markley，J.L.，和Lutsenko，S.（2006）威尔逊病蛋白质的N-结构域的溶液结构：独特的核苷酸结合环境和疾病突变的影响Proc. Natl. Acad. Sci.联合S. A 103，5302-5307。 2. Lutsenko，S.，叶夫列莫夫河G.，齐夫科夫斯基河，和步行者，J.M.（2002）人类铜转运ATP酶ATP 7 B（威尔逊病蛋白）：生物化学特性和调节J. Bioenerg。Biomembr. 34，351-362.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this project is to solve the structures of the nucleotide binding domains of the two human copper-transporting ATPases, Wilson disease associated protein and Menkes disease associated protein and to elucidate conformational changes caused in the domains by ATP binding. The copper-transporting ATPases belong to a distinct and poorly studied class of membrane transport enzymes (Lutsenko et al., 2002) and are essential for copper-metabolism in human tissues. Mutations in the ATP7A and ATP7B genes encoding these proteins result in severe hereditary metabolic disorders. No high-resolution structure of a heavy metal transporting ATPase has been published to date.The structures of the nucleotide binding domains will be solved in the nucleotide-free and ATP-bound forms. Location of the ATP -binding site in the three-dimensional structure of the domains will be determined. Conformational changes taking place upon ATP binding will be studied in the context of the catalytic cycle of the P-type ATPases, with the emphasis on unique features separating heavy-metal transporting ATPases from well studied Ca2+-ATPase. The differences in the three-dimensional structures of the nucleotide binding domains of Wilson disease and Menkes disease proteins associated with non-homologous regions of primary structure will be determined. This project will also provide groundwork for the future studies of the structural effects of disease-causing mutations in the copper-transporting ATPases, for solving the structures of the larger domains of Wilson and Menkes disease proteins and mapping the domain-domain interaction surfaces. To date the structure of the N-nucleotide-binding domain of the Wilson disease ATPase (WNABD) comprising residues 1036-1196 has been solved in ATP bound form (Dmitriev et al., 2006). Data collection and processing for structure determination of two disease-causing mutant variants of the N-domain (E1064A and H1069A) is in progressReferences 1. Dmitriev, O., Tsivkovskii, R., Abildgaard, F., Morgan, C. T., Markley, J. L., and Lutsenko, S. (2006) Solution structure of the N-domain of Wilson disease protein: Distinct nucleotide-binding environment and effects of disease mutations Proc. Natl. Acad. Sci. U. S. A 103, 5302-5307. 2. Lutsenko, S., Efremov, R. G., Tsivkovskii, R., and Walker, J. M. (2002) Human copper-transporting ATPase ATP7B (the Wilson's disease protein): biochemical properties and regulation J. Bioenerg. Biomembr. 34, 351-362.