DETERMINATION OF 3D STRUCTURE OF A P-TYPE ATPASE

P 型ATP酶 3D 结构的测定

• 批准号: 6602937
• 负责人: Dinesh A Yernool
• 金额: $ 2.99万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6602937
• 关键词: Escherichia coli; X ray crystallography; detergents; enzyme activity; enzyme complex; enzyme model; enzyme structure; hydrogen transporting ATP synthase; immunoglobulin structure; membrane proteins; protein purification

The overall objective of the proposal is to determine the 3D structure of a P-type ATPase, specifically the Kdp-ATPase from E. coli. P-type ATPases are membrane bound active transporters involved in ion homeostasis in all forms of life. Although, extensive work has been reported on the biochemistry and molecular biology of P-type ATPases, the molecular mechanisms of ion translocation and energy transduction remain unknown due to the lack of atomic resolution structures. This proposal is directed towards filling this gap in knowledge. X-ray crystallographic methods will be used to determine the structure of Kdp-ATPase, a high affinity K+ transporter. Kdp-ATPase from E. coli was chosen for structural studies as large quantities of protein can be expressed and purified easily as a multi-subunit complex. In addition, its biochemical properties are well characterized and a number of mutants are available. However, to overcome the difficulties associated with crystallography of membrane proteins, a multi-pronged approach involving crystallization of Kdp-ATPase with detergents, complexed to antibody fragments, bound to inhibitors and in lipidic cubic phases, are proposed. Besides enhancing our understanding of the Kdp complex, the 3D structure will elucidate the functioning of related eukaryotic ATPases including the human sarcoplasmic Ca2+ ATPase. Further, homologs of Kdp-ATPase are found only among eubacteria including many pathogenic bacteria. The availability of a high-resolution structure will be a start point for rational drug design efforts.

该提案的总体目标是确定P型ATPase的3D结构，特别是来自大肠杆菌的KDP-ATPase。P型ATPase是一种膜结合的活性转运蛋白，参与所有生命形式的离子动态平衡。尽管P型ATPase的生物化学和分子生物学已有大量研究报道，但由于缺乏原子分辨结构，离子转运和能量转导的分子机制尚不清楚。这项建议旨在填补这一知识空白。X射线结晶学方法将用于确定KDP-ATPase的结构，KDP-ATPase是一种高亲和力的K+转运体。由于KDP-ATPase能够以多亚基复合体的形式大量表达和纯化，因此我们选择从E.ColiKDP-ATPase中进行结构研究。此外，它的生化特性得到了很好的表征，并有许多突变体可用。然而，为了克服与膜蛋白结晶学相关的困难，人们提出了一种多管齐下的方法，包括KDP-ATPase与去污剂结晶、与抗体片段络合、与抑制剂结合以及在脂类立方相中结晶。除了加深我们对KDP复合体的理解外，3D结构还将阐明相关的真核ATPase的功能，包括人肌浆钙ATPase。此外，KDP-ATPase的同源物只在包括许多病原菌在内的真细菌中被发现。高分辨率结构的可获得性将是合理药物设计努力的起点。