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

在真核生物中，钙离子作为细胞内信号转导的第二信使发挥着重要作用。窖内钙浓度的升高是由钙通道介导窖外钙的流入和内质网钙的释放引起的。钙结合蛋白与钙结合，在窖内钙升高时引起构象改变，修饰下游靶分子。CHP1由195个氨基酸组成，与calcalineurin （CNB）的调控B亚基具有很大的序列相似性。从氨基酸序列来看，CHP1有4个ef -手（EF-1-4），在其n端被称为钙结合基序和n -肉豆蔻酰化基序。CHP1还被鉴定为与Na+/H+交换器1 （NHE1）的窖内近膜区相互作用的蛋白。CHP1是NHEls活性所必需的，CHP1结合缺陷突变体NHE1表现出明显的窖内ph的酸性转移。此外，缺乏钙结合亲和力的突变体Na+/H+交换活性也显著降低。此外，最近的研究表明，CHP1与DAPK相关的凋亡诱导激酶2 （DRAK2）结合并抑制其激酶活性。CHP1显著降低了DRAK2对自身磷酸化和外源底物磷酸化的激酶活性（抑制约85%）。DRAK2是一种由371个氨基酸组成的丝氨酸/苏氨酸激酶，主要由激酶结构域（残基33-293）组成。DAP激酶家族的一些成员的酶活性是通过钙调蛋白与钙调蛋白结合区域的结合来调节的。有趣的是，钙和CHP1负向调节DRAK2的激酶活性，这是诱导凋亡所必需的。NHE1和DRAK2的CHP1结合区域相似性较低，因此CHP1可能以多种方式与多个靶点相互作用。尽管CHP1的多种细胞内功能非常重要，但尚未获得CHP1的结构信息。在本研究中，我们对CHP1进行了x射线晶体学分析，以阐明CHP1的靶识别机制。我们还进行了DRAK2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