Mechanisms of Ion Selection in P-type ATPases

P 型 ATP 酶中的离子选择机制

• 批准号: 6371101
• 负责人: RAJINI RAO
• 金额: $ 28.55万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6371101
• 关键词: Golgi apparatus; bioenergetics; calcium; calcium transporting ATPase; cell membrane; chemical kinetics; computer simulation; conformation; fungal genetics; fungal proteins; intermolecular interaction; ion transport; manganese; model design /development; molecular dynamics; molecular site; physical model; protein folding; protein purification; protein structure function; radiotracer; scintillation counter; site directed mutagenesis; structural biology; suppressor mutations; yeasts

Many important cellular and physiological events, including nutrient uptake, signal transduction and cell cycle progression are mediated by transmembrane ion gradients. An extensive, multigene family of cation pumps, the P-ATPases, have evolved to transport a wide variety of different ions (Ca2+, Na+, K+, H+, Mg2+, Cu2+, to name a few). In keeping with their essential roles, the P-ATPases are a target for pharmacological intervention in disease (such as congestive heart failure and stomach ulcers), and are defective in various inherited disorders (Menkes, Wilson, Brody and Hailey-Hailey disease). Despite the similarities in sequence, structure and mechanism within this family, individual members differ strikingly in ion selectivity. The molecular basis of selectivity in ion pumps remains one of the fundamental unanswered problems in the field of membrane bioenergetics. To approach this problem, we will: focus on the Golgi Ca2+, Mn2+-ATPase, Pmrl, in the genetically tractable organism yeast, apply simple and powerful phenotypic screens that will identify loss of function or selectivity mutations, develop rigorous biochemical tools to analyze the defective pumps. In Aim 1, we will identify the molecular determinants of divalent cation selectivity in yeast Pmrl, a founding member of the newly- defined subgroup of Golgi/secretory pathway Ca2+-ATPases. Specifically, we will focus on selectivity for Mn2+ versus Ca2+ ions. In one approach, we will use directed and random mutagenesis techniques in conjunction with biological assays for Ca2+ chelator and Mn2+ toxicity to identify mutations that alter ion selectivity. In a second approach, we will use homology modeling of yeast Pmrl, based on the known crystal structure of the SERCA pump, to design rational targets for mutagenesis. Target residues will include those predicted to line the ion conducting pathway, stabilize adjacent membrane helices or form domain interfaces. In Aim 2, loss-of-function mutants with interesting properties such as alterations in ion selectivity or uncoupling of ATPase hydrolysis from ion transport, will be further mutagenized and subjected to phenotypic selection in order to identify intragenic suppressor mutations. These will provide unique insight on critical interactions between domains, and within or between membrane helices, that will complement structural information on ion pumps. In Aim 3, large-scale purification of Pmrl from fermentor-grown Pichia pastoris cultures will be undertaken for structural studies on cation binding and the concomitant conformational changes. Taken together, these aims constitute a powerful approach toward deciphering the molecular basis of selectivity and transport in ion pumps.

跨膜离子梯度介导了许多重要的细胞和生理事件，包括营养摄取，信号转导和细胞周期进程。阳离子泵的广泛多基因家族，P-ATPases已演变为运输各种不同的离子（Ca2+，Na+，K+，H+，Mg2+，Cu2+，仅举几例）。 为了保持其基本作用，P-ATP酶是对疾病的药理干预（例如充血性心力衰竭和胃溃疡）的目标，并且在各种遗传性疾病（Menkes，Wilson，Brody，Brody和Hailey-Hailey病）中有缺陷。 尽管该家族内的序列，结构和机制的相似性，但个体成员在离子选择性方面有明显的不同。离子泵中选择性的分子基础仍然是膜生物能学领域的基本未解决问题之一。 为了解决这个问题，我们将：专注于Golgi Ca2+，Mn2+-ATPase，PMRL，在遗传上可牵引的生物体酵母中，应用简单而强大的表型筛选，这些筛选将识别功能或选择性突变的丧失，开发严格的生物化学工具来分析缺陷泵。在AIM 1中，我们将确定酵母PMRL中二价阳离子选择性的分子决定因素，酵母PMRL是Golgi/分泌途径Ca2+-ATPases的新定义亚组的创始成员。具体而言，我们将专注于MN2+与Ca2+离子的选择性。 在一种方法中，我们将使用定向和随机的诱变技术与CA2+螯合剂和MN2+毒性的生物测定法结合使用，以识别改变离子选择性的突变。 在第二种方法中，我们将根据SERCA泵的已知晶体结构对酵母PMRL的同源性建模来设计诱变的合理靶标。目标残留物将包括预测将离子导电途径排列，稳定相邻膜螺旋或形成域界面的那些。 在AIM 2中，具有有趣特性的功能丧失突变体，例如离子选择性的改变或从离子转运的ATPase水解解偶联的变化，将进一步诱变并接受表型选择，以识别基因内抑制器突变。 这些将为域之间以及膜内或之间的关键相互作用提供独特的见解，这些互动将补充有关离子泵的结构信息。 在AIM 3中，将对阳离子结合和伴随的构象变化进行大规模纯化PMRL的大规模纯化。 综上所述，这些目标构成了一种强大的方法来破译离子泵中选择性和运输的分子基础。