Structural study for the recognition mechanism of the target protein by a novel calcium sensor

钙离子是真核生物细胞内信号转导的第二信使。细胞内钙离子浓度升高是由钙通道介导的细胞外钙离子内流和内质网释放钙引起的。钙结合蛋白与钙结合，并在细胞内钙升高时引起构象变化以修饰下游靶分子。CHP 1由195个氨基酸组成，与钙调神经磷酸酶（CN B）的调节B亚基显示出实质性的序列相似性。基于氨基酸序列，CHP 1具有4个EF-手（EF-1-4），其被称为钙结合基序和在其N-末端的N-豆蔻酰化基序。CHP 1也被鉴定为与Na+/H+交换器1（NHE 1）的细胞内近膜区相互作用的蛋白。CHP 1是NHE 1活性所必需的，CHP 1结合缺陷的NHE 1突变体显示出细胞内pH的显著酸性移动。 关于我们 y，也具有显著降低的Na+/H+交换活性。近年来的研究表明，CHP 1与DAPK相关的凋亡诱导激酶2（DRAK 2）结合，抑制其激酶活性。CHP 1显著降低（约85%抑制）DRAK 2的自磷酸化和外源底物磷酸化的激酶活性。DRAK 2是由371个氨基酸组成的丝氨酸/苏氨酸激酶，主要由激酶结构域组成（残基33-293）DAP激酶家族的一些成员的酶活性通过钙调蛋白与钙调蛋白结合区的结合来调节。有趣的是，钙和CHP 1负性调节细胞凋亡诱导所需的DRAK 2的激酶活性。NHE 1和DRAK 2的CHP 1结合区具有低的相似性，因此CHP 1可以以多种方式与多个靶标相互作用。尽管CHP 1的多种细胞内功能非常重要，但尚未获得CHP 1的结构信息。在这项研究中，我们进行了X射线晶体学分析的CHP 1，以澄清CHP 1的目标识别机制。我们还进行DRAK 2的结晶。少

Calcium ion plays significant role as a second messenger of intra cellular signaling in eukaryote. Rising concentration of intra cellar calcium is arisen by afflux of extra cellar calcium mediated by calcium channel and release of calcium from endoplasmic reticulum. Calcium binding protein binds to calcium and causes conformation change to modify down stream target molecules when intra cellar calcium rises.CHP1 is comprised of 195 amino acids and shows substantial sequence similarity to the regulatory B subunit of calcineurin (CNB). Based on amino acid sequence CHP1 has 4 EF-hands (EF-1-4) that is known as calcium binding motif and N-myristoylation motif on its N-terminal..CHP1 was also identified as a protein that interacts with intra cellar juxta-membrane region of Na+/H+ exchanger 1 (NHE1). CHP1 is essential for NHEls activity, and CHP1 binding defective mutant of NHE1 show a marked acidic shift of intra cellar pH. Furthermore, a mutant which displays lacking calcium-binding affinit … More y, also has a significantly reduced Na+/H+ exchange activity. Furthermore recent study reveals that CHP1 binds to DAPK related apoptosis inducing kinase 2 (DRAK2) and inhibits its kinase activity. CHP1 significantly reduced (〜85% inhibition) the kinase activity of DRAK2 for both autophosphorylation and phsphorylation of exogenous substrate. DRAK2 is a Ser/Thr kinase consisting of 371 amino acids and mainly consists of kinase domain (residues 33-293) The enzyme activity of some members of DAP kinase family is regulated via binding of calmodulin to a calmodulin binding region It is interesting that calcium and CHP1 negatively regulate the kinase activity of DRAK2 required for apoptotic induction.CHP1 binding regions of NHE1 and DRAK2 have low similarity, so CHP1 may interact with multiple target on multiple manner. Despite the great importance of multiple intracellular functions of CHP1, no structural information of CHP1 has been obtained. In this study we performed X-ray crystallographic analysis of CHP1 to clarify CHP1s target recognition mechanism. We also perform crystallization of DRAK2. Less