Ca2+ Transport Mechanism of CaCA Protein Family

CaCA蛋白家族的Ca2+转运机制

基本信息

批准号：
8085646
负责人：
Lei Zheng
金额：
$ 28.5万
依托单位：
UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-04-01 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8085646
关键词：
Address Anions Bacillus subtilis Back Biochemical Biological Biological Assay Biological Process Cardiac Cardiovascular Diseases Carrier Proteins Cations Cell membrane Cell physiology Cells Cellular Membrane Charge Coupled Coupling Crystallization Crystallography Data Dependence Drosophila genus Drug Design Electrophysiology (science)Family Family member Functional disorder Goals Heart failure Homeostasis Homologous Gene Hormones Human Hypertension In Vitro Investigation Ion Transport Ions Light Mammals Measures Mediating Membrane Membrane Proteins Molecular Molecular Conformation Motion Mutagenesis Myocardium Neurons Pathology Pathway interactions Phosphorothioate Oligonucleotide Physiological Play Prokaryotic Cells Property Protein Family Proteins Protons Regulation Relaxation Resolution Role Sequence Analysis Signal Transduction Signal Transduction Pathway Site-Directed Mutagenesis Staging Stroke Structural Models Structure Subcellular structure Surface Synaptic Transmission Testing Tissues Transmembrane Domain Vesicle Wing antiporter aptamer base design innovation inorganic phosphate insight member neurotransmission novel symporter

项目摘要

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. PUBLIC HEALTH RELEVANCE: Ca2+ transport across the cell membrane is essential for many important biological activities including cardiac contraction, neural transmission and hormone secretion. Our studies focus on an important Ca2+ transport protein family, the CaCA proteins. These proteins extrude Ca2+ from cells to outside, helping the Ca2+-excited cells such as cardiac muscle back to the relaxation stage. Dysfunction of the proteins results in many cardiovascular diseases including heart failure, stroke and Na+-dependent hypertension. Our project is to address functional questions regarding the Ca2+ transport and the regulatory mechanism of this protein family. We aim to understand how the Ca2+ extrusion is carried out by the protein and how the Ca2+ efflux activity is regulated using a combination of x-ray crystallography, biophysical and biochemical approaches. Our proposed studies will provide important information to understand pathology of related cardiovascular diseases and facilitate specific drug design.

描述（由申请人提供）：Ca2+/阳离子反向转运蛋白（CaCA）是膜质中主要的次级 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，包括哺乳动物 CaCA 蛋白中的心脏收缩和神经元传递。 Ca2+ 通过其跨膜结构域的转运受到其细胞内调节结构域的严格控制，然而，由于缺乏 CaCA 蛋白的原子结构，人们对 Ca2+ 转运和 CaCA 蛋白调节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且相互关联的机制：1）为了阐明Ca2+转运机制，我们结晶了YfkE蛋白，一种原核Ca2+转运蛋白和CaCA同源物共享保守的。哺乳动物 CaCA 的膜拓扑和序列我们已经获得了衍射至 6 ¿ YfkE 蛋白的原子结构不仅为分析 CaCAs 的 Ca2+ 转运机制提供了第一个结构基础，而且还提供了第一个机会。从结构角度理解 Ca2+ 选择性和电导率对于 Ca2+ 稳态至关重要；2) 我们发现 YfkE 蛋白的 Ca2+ 转运活性与磷酸根阴离子共转运相结合，我们将通过内向外囊泡中的诱变来分析 Ca2+/磷酸盐共转运途径。我们将测试 Ca2+/磷酸盐共转运是否发生在其他 CaCA 蛋白中。 3) 为了阐明哺乳动物 CaCA 蛋白的调节机制，我们进行了初步结构研究果蝇CaCA蛋白CALX表明，这种调节是通过细胞内调节域中Ca2+和Na+相互作用诱导的子域构象变化来实现的。为了检验这一假设，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检查调节机制。此外，通过结合原核Ca2+转运蛋白YfkE和真核CALX调节域的结构，我们将能够生成第一个结构模型来理解重要 CaCA 蛋白的 Ca2+ 转运和调节机制。公共健康相关性：Ca2+ 跨细胞膜转运对于许多重要的生物活动至关重要，包括心脏收缩、神经传递和激素分泌。我们的研究重点是重要的 Ca2+ 转运蛋白家族，即 CaCA 蛋白，这些蛋白质将 Ca2+ 从细胞排出到外部。，帮助 Ca2+ 兴奋的细胞（例如心肌）恢复到松弛阶段。蛋白质功能障碍会导致许多心血管疾病，包括心力衰竭、中风和 Na+ 依赖性高血压。我们旨在了解该蛋白质如何进行 Ca2+ 挤出以及如何结合 X 射线晶体学、生物物理和生化方法来调节 Ca2+ 流出活性。我们提出的研究将为了解相关心血管疾病的病理学并促进具体的药物设计提供重要信息。