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

肌浆网Ca^<2+>-ATP酶（SERCA 1a）催化Ca^<2+>转运和ATP水解。我们以前的研究表明，A结构域的大旋转及其与P和N结构域的紧密接触很可能发生在磷酸化中间体的异构转变过程中（E1PCa_2向E2PCa_2转变）和Ca^<2 +>释放形成最致密的E2 P释放形式的单个头片段，并认为E2 P中3个胞质结构域之间的紧密接触所提供的稳定能量将为跨膜螺旋的移动和结合的Ca^<2+>释放到管腔中提供能量。本研究通过定点突变和动力学分析进一步探索了这些结构域的运动和相互作用的区域和残基，发现：1. Glu^<40>-Ser^<48>环具有合适的长度（连接A结构域和第一跨膜螺旋（M1））在E1PCa_2到E2PCa_2的过程中对A结构域的大旋转和M1的协调运动至关重要。2 transition.2.Arg334在M4的顶部（连接到P结构域）和M2顶部的Tyr 122（与A结构域相连）分别在E1PCa_2向E2PCa_2的转变以及随后的Ca^&lt;2+&gt;释放形式和E2 P的水解中起重要作用，在E2 P中，A结构域的最外层Val 200环是A结构域和P结构域紧密接触的区域之一我们还开发了稳定的E2 P类似物，以及溶解、纯化和稳定该类似物以用于结晶的条件。我们还揭示了导致Daner病的A结构域Δ41，Δ42，N39 D和N39 T突变对动力学性质的影响，以了解疾病的分子机制。

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.