STRUCTURE OF THE SODIUM PUMP NUCLEOTIDE BINDING DOMAIN

描述：(逐字摘自申请人的摘要)钠泵已被 充血性心力衰竭的治疗靶点 强心苷已经有一个多世纪了。不幸的是，仍然没有什么 了解心脏糖苷抑制的机制。实验研究 在过去的三十年里建立了一些关系， 钠泵催化的生化反应和传输反应 它起到了调解作用。然而，ATP水解酶的分子机制 与离子的“上行”运动相耦合仍然是一个谜。这很可能是 源于这样一个事实，即人们对 该蛋白的结构-功能关系。我们的长期目标是获得 完全了解钠泵的传输机制，这将 需要各种类型的酶的高分辨率X射线衍射图 构象与特定氨基酸残基的鉴定 与配基结合有关的。然而，获得质量所需的技术 膜蛋白的结晶还不够完善。具体的 这项提案中概述的实验将利用细菌遗传学来 过表达Na，K-ATPase的大细胞质环(目标I)。所有的 到目前为止，与ATP结合和水解有关的残基已在 这部分蛋白质。同样的道理也适用于本组织的所有成员 重要的蛋白质家族(即P型ATPase)。三磷酸腺苷结合 野生型和突变型三磷酸腺苷结合区的特征如下 CD和X-射线结晶学的确定和结构表征 技术(目标三)。此外，我们将确定其他哪些部分 Na，K-ATPase在物理上与ATP结合域相互作用(AIM II)。这个 这项工作的结果将提供一张参与ATP的氨基酸图谱 从而有助于阐明ATP之间的偶联机制 水解性和阳离子转运。钠泵在各种情况下都是至关重要的 维持体液和电解质平衡的器官。这些过程是动态的 平衡和这种平衡可能在多种疾病中被破坏 各州。在对这些病理情况进行充分描述之前 因此，需要对钠泵的功能有更全面的了解。

DESCRIPTION: (Verbatim from the Applicant's Abstract) The sodium pump has been the target for the therapeutic treatment of congestive heart failure with cardiac glycosides for more than a century. Unfortunately there is still little known about the mechanism of cardiac glycoside inhibition. Experimental studies over the past three decades have established some relationships between the biochemical reactions catalyzed by the sodium pump and the transport reactions it mediates. However, the molecular mechanisms by which the hydrolysis of ATP is coupled to the "uphill" movement of ions remains a mystery. This probably stems from the fact that there remains little known about the structure-function relationship of this protein. Our long term goal is to gain a complete understanding of the sodium pump transport mechanism which will require high-resolution x-ray diffraction patterns of the enzyme in its various conformations and the identification of specific amino acids residues associated with ligand binding. However, the techniques required to get quality crystals of membrane proteins have not yet been perfected. The specific experiments outlined in this proposal will exploit bacterial genetics to overexpress the large cytoplasmic loop of the Na,K-ATPase (Aim I). All the residues thus far implicated in ATP binding and hydrolysis have been found in this section of the protein. The same holds true for all members of this important protein family (i.e., P-type ATPases). The ATP binding characteristics of the wild type and mutant ATP binding domains will be determined and structurally characterized using CD and x-ray crystallographic techniques (Aim III). In addition, we will determine which other sections of the Na,K-ATPase physically interact with the ATP binding domain (Aim II). The results from this work will provide a map of the amino acids involved in ATP coordination and thus help elucidate the coupling mechanism between ATP hydrolysis and cation transport. The sodium pump is vital in a variety of organs for fluid and electrolyte balance. These processes are in a dynamic equilibrium and this equilibrium can become disrupted in a variety of disease states. Before an adequate description of these pathological situations can be made, a more complete understanding of sodium pump function is required.