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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 本项目的目的是解决人类两种铜转运ATPase，威尔逊病相关蛋白和门克斯病相关蛋白的核苷酸结合域的结构，并阐明ATP结合在这些结合域中引起的构象变化。铜转运ATPase属于一类独特的膜转运酶(Lutsenko et al.，2002)，对人体组织中的铜代谢是必不可少的。编码这些蛋白质的ATP7A和ATP7B基因突变会导致严重的遗传性代谢紊乱。到目前为止，还没有关于重金属转运ATPase的高分辨结构的报道，核苷酸结合区的结构将以无核苷酸和ATP结合的形式求解。将确定ATP结合位点在结构域的三维结构中的位置。结合ATP时发生的构象变化将在P型ATPase催化循环的背景下进行研究，重点是区分重金属转运ATPase和已有研究的Ca2+-ATPase的独特特征。将确定肝豆状核变性和门克斯病蛋白质核苷酸结合域的三维结构与初级结构的非同源区域的差异。该项目还将为未来研究铜转运ATPase致病突变的结构效应，为解决Wilson和Menkes病蛋白更大结构域的结构和绘制结构域间相互作用面提供基础。到目前为止，由1036-1196残基组成的Wilson病ATPase(WNABD)的N-核苷酸结合域的结构已经以ATP结合的形式解决(Dmitriev等人，2006年)。用于确定两个致病N区突变变体(E1064A和H1069A)的数据收集和处理正在进行中参考文献1.Dmitriev，O.，Tsivkovskii，R.，Abildgaard，F.，Morgan，C.T.，Markley，J.L.和Lutsenko，S.(2006)威尔逊病蛋白N区的解决方案结构：不同的核苷酸结合环境和疾病突变过程的影响。娜塔莉。阿卡德。SCI。美国A 103,5302-5307。2.Lutsenko，S.，Efemov，R.G.，Tsivkovskii，R.和Walker，J.M.(2002)人类铜转运ATP7B(威尔逊病蛋白)：生化特性和调控J.Biomembr34,351-362

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.