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Structure and Function of Sarco(endo)plasmic Reticulum Ca^<2+>-ATPase and Mechanism of Darier's Diseases Caused by its Abnormality

肌(内)质网Ca^2-ATP酶的结构与功能及其异常引起达里尔病的机制

基本信息

批准号：
14580639
负责人：
SUZUKI Hiroshi
金额：
$ 1.98万
依托单位：
Asahikawa Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

Sarcoplasmic reticulum Ca^<2+>-ATPase (SERCA1a) catalyzes Ca^<2+> transport coupled with ATP hydrolysis. We previously showed that the large rotation of A domain and its intimate contact with P and N domains most likely occur during the isomeric transition of phosphorylated intermediate(E1PCa_2 to E2PCa_2 transition) and Ca^<2+> release to form the most compactly organized single headpiece in the Ca^<2+>-released form of E2P, and suggested that stabilization energy provided by the intimate contacts between 3 cytoplasmic domains in E2P will provide energy for moving transmembrane helices and release the bound Ca^<2+> into lumen. In this research, we further explored the regions and residues responsible for the movements of and interactions between the domains by site-directed mutagenesis and kinetic analysis with the mutants, and found follows.1.The Glu^<40>-Ser^<48> loop with its proper length(connecting A domain and 1st transmembrane helix(M1)) is critical for the large rotation of A domain and coordinated motion of M1 during the E1PCa_2 to E2PCa_2 transition.2.Arg334 on the top of M4(connected to P domain)and Tyr122 on the top of M2(connected to A domain)play essential roles in the E1PCa_2 to E2PCa_2 transition and in the subsequent Ca^<2+> release form and hydrolysis of E2P, respectively, by contributing to the motions of and interactions between A and P domains.3.The outermost Val200 loop of A domain is one of the regions responsible for the intimate contact between A and P domains in E2P to release bound Ca^<2+> into lumen.We also developed the stable E2P analogue and conditions to solubilize, purify, and stabilize the analogue for the crystallization. We also revealed the effects of mutations on A domain responsible for Daner's disease, Δ41,Δ42,N39D, and N39T on the kinetic properties in order to understand the molecular mechanism of the disease.

肌浆网Ca^<2+>-ATP酶（SERCA1a）催化Ca^<2+>转运并偶联ATP水解。我们之前的研究表明，A结构域的大旋转及其与P和N结构域的密切接触最有可能发生在磷酸化中间体的异构转变（E1PCa_2到E2PCa_2的转变）和Ca^<2+>的释放过程中，以形成Ca^<2+>-释放形式中组织最紧密的单个头状结构。表明E2P中3个细胞质结构域之间的密切接触所提供的稳定能量将为跨膜螺旋的移动提供能量，并将结合的Ca^<2+>释放到腔内。在本研究中，我们通过位点定向诱变和与突变体的动力学分析，进一步探索了负责结构域运动和相互作用的区域和残基，发现如下：1。在E1PCa_2向E2PCa_2转变过程中，长度合适的Glu^<40>-Ser^<48>环（连接A结构域和第一跨膜螺旋（M1））对于A结构域的大旋转和M1的协调运动至关重要。位于M4顶端的Arg334（连接到P结构域）和位于M2顶端的Tyr122（连接到A结构域）分别通过促进A和P结构域的运动和相互作用，在E1PCa_2到E2PCa_2的转变和随后的Ca^<2+>的释放形式和E2P的水解中起重要作用。A结构域最外层的Val200环是E2P中负责A和P结构域密切接触的区域之一，将结合的Ca^<2+>释放到管腔中。我们还开发了稳定的E2P类似物以及溶解、纯化和稳定结晶类似物的条件。我们还揭示了Daner病A结构域（Δ41，Δ42，N39D和N39T）突变对动力学特性的影响，以了解疾病的分子机制。