Jain Ruchi, Kumar Shivesh, Gourinath Samudrala, Bhattacharya Sudha, Bhattacharya Alok
School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
PLoS One. 2009;4(4):e5269. doi: 10.1371/journal.pone.0005269. Epub 2009 Apr 22.
Entamoeba histolytica, a protozoan parasite, is the causative agent of amoebiasis, and calcium signaling is thought to be involved in amoebic pathogenesis. EhCaBP1, a Ca(2+) binding protein of E. histolytica, is essential for parasite growth. High resolution crystal structure of EhCaBP1 suggested an unusual arrangement of the EF-hand domains in the N-terminal part of the structure, while C-terminal part of the protein was not traced. The structure revealed a trimer with amino terminal domains of the three molecules interacting in a head-to-tail manner forming an assembled domain at the interface with EF1 and EF2 motifs of different molecules coming close to each other. In order to understand the specific roles of the two domains of EhCaBP1, the molecule was divided into two halves, and each half was separately expressed. The domains were characterized with respect to their structure, as well as specific functional features, such as ability to activate kinase and bind actin. The domains were also expressed in E. histolytica cells along with green fluorescent protein. The results suggest that the N-terminal domain retains some of the properties, such as localization in phagocytic cups and activation of kinase. Crystal structure of EhCaBP1 with Phenylalanine revealed that the assembled domains, which are similar to Calmodulin N-terminal domain, bind to Phenylalanine revealing the binding mode to the target proteins. The C-terminal domain did not show any of the activities tested. However, over-expression in amebic cells led to a dominant negative phenotype. The results suggest that the two domains of EhCaBP1 are functionally and structurally different from each other. Both the domains are required for structural stability and full range of functional diversity.
溶组织内阿米巴是一种原生动物寄生虫，是阿米巴病的病原体，钙信号传导被认为与阿米巴发病机制有关。EhCaBP1是溶组织内阿米巴的一种钙结合蛋白，对寄生虫生长至关重要。EhCaBP1的高分辨率晶体结构表明，在该结构的N端部分，EF手型结构域排列异常，而该蛋白的C端部分未被追踪到。该结构显示，三个分子的氨基末端结构域以头对尾的方式相互作用，在界面处形成一个组装结构域，不同分子的EF1和EF2基序彼此靠近。为了了解EhCaBP1两个结构域的具体作用，将该分子分成两半，并分别表达每一半。对这些结构域的结构以及特定功能特征进行了表征，例如激活激酶和结合肌动蛋白的能力。这些结构域还与绿色荧光蛋白一起在溶组织内阿米巴细胞中表达。结果表明，N端结构域保留了一些特性，如定位在吞噬杯中以及激活激酶。EhCaBP1与苯丙氨酸的晶体结构表明，与钙调蛋白N端结构域相似的组装结构域与苯丙氨酸结合，揭示了与靶蛋白的结合模式。C端结构域未表现出任何测试的活性。然而，在阿米巴细胞中过表达导致显性负性表型。结果表明，EhCaBP1的两个结构域在功能和结构上彼此不同。两个结构域对于结构稳定性和功能多样性的全面发挥都是必需的。
