Ca2+ Transport Mechanism of CaCA Protein Family

描述（由申请人提供）：Ca2+/阳离子反转运蛋白（CaCAs）是质膜中主要的次级Ca2+转运蛋白。它们在许多重要的Ca2+介导的生物过程中发挥重要作用，包括心脏收缩和神经元传递。在哺乳动物CaCA蛋白中，Ca2+通过其跨膜结构域的运输受到其细胞内调节结构域的严格控制。然而，由于缺乏任何家族成员的原子结构，人们对Ca2+运输和CaCA蛋白调控的分子机制知之甚少。我们设计了一种策略，通过结构和功能研究来阐明这两个重要的相互关联的机制：1)为了阐明Ca2+转运机制，我们结晶了YfkE蛋白，这是一种原核Ca2+转运蛋白和CaCA同源物，与哺乳动物的CaCA共享保守的膜拓扑结构和序列。我们已经获得了衍射到6°分辨率的晶体，并设计了创新的方法来优化x射线晶体学结构测定的结晶。YfkE蛋白的原子结构不仅为分析Ca2+转运机制提供了第一个结构基础，而且为从结构上理解Ca2+稳态所必需的Ca2+选择性和电导率提供了第一个机会；2)我们发现，YfkE蛋白的Ca2+转运活性与磷酸阴离子共转运偶联，这是CaCA机制的一个以前未被认识的方面。我们将分析Ca2+/磷酸盐共运输途径突变在内外囊泡。我们将测试是否Ca2+/磷酸共运输发生在其他CaCA蛋白。这些研究将提供Ca2+/磷酸盐共运输的第一个数据，并提供磷酸盐参与Ca2+稳态的见解；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Ca2+和Na+相互作用引起的亚结构域构象变化来实现的。为了验证这一假设，我们将确定CALX细胞内结构域的结构，并通过诱变和电生理学研究其调控机制。此外，通过结合原核Ca2+转运体YfkE和真核CALX调节结构域的结构，我们将能够生成第一个结构模型来理解Ca2+转运和重要的CaCA蛋白的调节机制。

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.