Ca2+ Transport Mechanism of CaCA Protein Family

描述(申请人提供)：钙离子/阳离子逆向转运蛋白(CAAs)是质膜上主要的次级钙离子转运蛋白。它们在许多重要的钙离子介导的生物过程中发挥着重要的作用，包括心脏收缩和神经元传递。在哺乳动物CACA蛋白中，钙离子通过其跨膜区的运输受到其细胞内调节域的严格控制。然而，由于该家族中没有任何成员的原子结构，因此对CaCA蛋白潜在的钙转运和调控的分子机制知之甚少。我们设计了一种策略，通过结构和功能的研究来阐明这两个相互关联的重要机制：1)为了阐明钙离子的转运机制，我们结晶了原核生物钙离子转运蛋白Yfke蛋白，它是一个与哺乳动物CACAs具有保守的膜拓扑结构和序列的CACA同源物。我们已经获得了分辨率为6°的晶体，并设计了创新的方法来优化结晶，用于X射线结晶学结构测定。Yfke蛋白的原子结构不仅为分析CaCA的钙转运机制提供了第一个结构基础，而且也为从结构上理解钙离子的选择性和钙稳态所必需的电导率提供了第一个机会；2)我们发现Yfke蛋白的钙转运活性与磷酸阴离子共转运耦合，这是以前未被认识的CACA机制的一个方面。我们将通过内向外小泡的诱变来分析钙/磷共转运途径。我们将测试在其他CACA蛋白中是否存在钙/磷共转运。这些研究将首次提供有关钙/磷共转运的数据，并为磷酸盐参与钙稳态提供洞察力；3)为了阐明哺乳动物CACA蛋白的调控机制，我们对果蝇CACA蛋白CALX的初步结构研究表明，这种调控是通过细胞内钙离子和钠离子相互作用引起的亚域构象变化实现的。为了验证这一假设，我们将确定Calx胞内结构域的结构，并通过诱变和电生理学检查其调控机制。此外，通过结合原核细胞钙离子转运蛋白Yfke的结构和真核细胞中Calx的调控结构域，我们将能够建立第一个结构模型来理解重要的CaCA蛋白的钙转运和调控机制。

DESCRIPTION (provided by applicant): Ca2+/cation antiporters (CaCAs) are the major secondary Ca2+ transporter proteins in the plasma membrane. They play essential roles in many important Ca2+-mediated biological processes including cardiac contraction and neuronal transmission. In mammalian CaCA proteins, Ca2+ transport through their transmembrane domain is tightly controlled by their intracellular regulatory domain. However molecular mechanisms underlying Ca2+ transport and regulation of CaCA proteins are poorly understood due to the absence of an atomic structure of any member of the family. We have designed a strategy to elucidate these two important and interrelated mechanisms by structural and functional studies: 1) to elucidate the Ca2+ transport mechanism, we have crystallized the YfkE protein, a prokaryotic Ca2+ transporter and CaCA homolog sharing conserved membrane topology and sequence with mammalian CaCAs. We have obtained crystals diffracting to 6 ¿ resolution, and have designed innovative approaches to optimize the crystallization for structure determination by x-ray crystallography. The atomic structure of YfkE protein will not only provide the first structural basis for analyzing the Ca2+ transport mechanism of CaCAs, but also offers the first opportunity to understand in structural terms the Ca2+ selectivity and conductivity essential for Ca2+ homeostasis; 2) We have found that the Ca2+ transport activity of the YfkE protein is coupled with phosphate anion co-transport, a previously unrecognized aspect of a CaCA mechanism. We will analyze the Ca2+/phosphate co- transport pathway by mutagenesis in inside-out vesicles. We will test whether Ca2+/phosphate co- transport occurs in other CaCA proteins. These studies will provide the first data on Ca2+/phosphate co- transport and provide insight to phosphate involvement in Ca2+ homeostasis; 3) to elucidate the regulatory mechanism of mammalian CaCA proteins, our preliminary structural studies with Drosophila CaCA protein CALX suggest that the regulation is achieved by subdomain conformational changes induced by Ca2+ and Na+ interactions in the intracellular regulatory domain. To test this hypothesis, we will determine structures of the intracellular domain of CALX and examine the regulatory mechanism by mutagenesis and electrophysiology. In addition, by combining the structures of the prokaryotic Ca2+ transporter YfkE and the eukaryotic regulatory domain of CALX, we will be able to generate the first structural model to understand Ca2+ transport and regulatory mechanisms of the important CaCA proteins.