Mechanisms of Ion Selection in P-type ATPases

许多重要的细胞和生理事件，包括营养吸收、信号转导和细胞周期进程，都是由跨膜离子梯度调节的。一个广泛的、多基因的阳离子泵家族，P-ATPase，已经进化为运输各种不同的离子(仅举几例)。与其基本功能一致，P-ATPase是药物干预疾病(如充血性心力衰竭和胃溃疡)的靶点，在各种遗传性疾病(Menkes、Wilson、Brody和Hailey-Hailey病)中存在缺陷。尽管该家族在序列、结构和机制上有相似之处，但个别成员在离子选择性方面存在显著差异。离子泵选择性的分子基础仍然是膜生物能量学领域中尚未回答的基本问题之一。为了解决这个问题，我们将：专注于遗传上易驯化的生物酵母中的高尔基钙、锰-ATPase、Pmr1，应用简单而强大的表型筛选来识别功能丧失或选择性突变，开发严格的生化工具来分析有缺陷的泵。在目标1中，我们将确定酵母Pmr1中二价阳离子选择性的分子决定因素，Pmr1是高尔基体/分泌途径钙-ATPase新亚组的创始成员。具体地说，我们将重点放在对Mn2+和Ca2+离子的选择性上。在一种方法中，我们将使用定向和随机突变技术，结合钙离子螯合剂和Mn2+毒性的生物测试来确定改变离子选择性的突变。在第二种方法中，我们将基于已知的SERCA泵的晶体结构，使用酵母Pmr1的同源建模来设计合理的突变靶标。靶残基将包括那些预计将排列在离子传导途径上、稳定相邻膜螺旋或形成结构域界面的残基。在目标2中，具有有趣特性的功能缺失突变体将被进一步诱变并进行表型选择，以确定基因内抑制突变。这些将提供关于结构域之间以及膜螺旋内部或之间的关键相互作用的独特见解，这将补充关于离子泵的结构信息。在目标3中，将从发酵罐培养的毕赤酵母培养物中大规模纯化Pmr1，用于阳离子结合及其伴随的构象变化的结构研究。综上所述，这些目标构成了破译离子泵选择性和传输的分子基础的有力途径。

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.