Ca2+ Transport Mechanism of CaCA Protein Family

描述（由申请方提供）：Ca 2 +/阳离子反向转运蛋白（CaCA）是质膜中的主要二级Ca 2+转运蛋白。它们在许多重要的Ca 2+介导的生物过程中发挥重要作用，包括心脏收缩和神经传递。在哺乳动物CaCA蛋白中，通过其跨膜结构域的Ca 2+转运受到其胞内调节结构域的严格控制。然而，由于缺乏该家族任何成员的原子结构，对Ca 2+转运和CaCA蛋白调节的分子机制知之甚少。我们设计了一个策略，以阐明这两个重要的和相互关联的机制，从结构和功能的研究：1）为了阐明钙转运机制，我们已经结晶YfkE蛋白，原核Ca 2+转运蛋白和CaCA同源物共享保守的膜拓扑结构和序列与哺乳动物CaCA。我们已经获得了衍射分辨率为6 ½的晶体，并设计了创新的方法来优化结晶，以通过X射线晶体学进行结构测定。YfkE蛋白的原子结构不仅为分析CaCAs的Ca ~（2+）转运机制提供了第一个结构基础，而且为从结构上理解Ca ~（2+）稳态所必需的Ca ~（2+）选择性和电导性提供了第一个机会; 2）我们发现YfkE蛋白的Ca ~（2+）转运活性与磷酸盐阴离子共转运偶联，这是CaCA机制的一个以前未被认识的方面。我们将通过在由内而外的囊泡中的诱变来分析Ca 2 +/磷酸盐共转运途径。我们将测试在其他CaCA蛋白中是否发生Ca 2 +/磷酸盐共转运。这些研究将提供关于Ca ~（2+）/磷酸盐共转运的第一批数据，并为磷酸盐参与Ca ~（2+）稳态提供见解; 3）为了阐明哺乳动物CaCA蛋白的调节机制，我们对果蝇CaCA蛋白CALX的初步结构研究表明，该调节是通过胞内调节结构域中Ca ~（2+）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的胞内结构域的结构，并通过诱变和电生理学研究其调节机制。此外，通过结合原核细胞Ca 2+转运蛋白YfkE和真核细胞CALX调控结构域的结构，我们将能够产生第一个结构模型，以了解Ca 2+转运和重要的CaCA蛋白的调控机制。

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.