Crystal Structure of Colicin E3

9728420 Shoham本研究的目的是确定大肠杆菌素E3与其抑制剂免疫蛋白复合物的晶体结构。 已经发现了三种合适的重原子衍生物，还将寻找更多。 对应于不对称单元中的四个分子的电子密度将被平均，以提高图的可解释性。 在完成结构测定后，将设计大肠杆菌素E3和免疫蛋白的位点特异性突变体，以探测某些残基对亲和力的贡献。 这些突变体将在体外和体内进行结构和功能表征。 大肠杆菌素E3是由大肠杆菌的某些菌株分泌的一种毒性蛋白。大肠杆菌，以消除生活在同一生态位的相关菌株的细菌。 大肠杆菌素E3具有三种功能：它与靶细胞包膜上的特异性受体结合，穿过敏感细胞的内膜内化到敏感细胞中，一旦进入细胞内，它通过在16 S核糖体RNA上切割特异性键来灭活受感染细胞的蛋白质生物合成机制。 因此，大肠杆菌素E3的毒性源于其酶活性。 这三种功能显然位于大肠杆菌素E3分子的三个不同结构域。 生产生物体通过免疫系统对大肠杆菌素E3的毒性具有免疫力。 大肠杆菌素E3是一种“免疫蛋白”，其以1：1的复合物与大肠杆菌素E3紧密结合，从而使其失活。 大肠杆菌素-免疫蛋白复合物是迄今为止观察到的最稳定的蛋白质-蛋白质复合物之一。 这项工作的目标是在分子水平上了解这些极其紧密的蛋白质-蛋白质相互作用的性质。 此外，晶体结构将提供有关免疫蛋白抑制机制的信息，以及可能参与催化的大肠杆菌素E3残基的鉴定。 酶-抑制剂相互作用的详细知识也将阐明切割位点附近核糖体的局部结构。 ***

9728420 Shoham The goal of this research is the crystal structure determination of colicin E3 in complex with its inhibitor, the immunity protein. Three suitable heavy-atom derivatives have already been found, and more will be searched for. The electron density corresponding to the four molecules in the asymmetric unit will be averaged in order to improve the interpretability of the map. Upon completion of the structure determination, site-specific mutants of colicin E3 and of the immunity protein will be designed, in order to probe the contribution of certain residues to the affinity. These mutants will be characterized both structurally and functionally in vitro as well as in vivo. Colicin E3 is a toxic protein secreted by certain strains of E. Coli in order to eliminate related strains of bacteria living in the same ecological niche. Colicin E3 carries out three functions: it binds to a specific receptor on the target cell envelope, is internalized into sensitive cells across their inner membrane, and once inside the cell, it inactivates the protein biosynthetic machinery of the infected cell by nicking a specific bond on 16S ribosomal RNA. The toxicity of colicin E3 thus stems from its enzymatic activity. The three functions are apparently localized on three different domains of the colicin E3 molecule. The producing organism is immune to the toxicity of colicin E3 by virtue of an "immunity protein" which tightly binds to colicin E3 in a 1:1 complex, and thus renders it inactive. Colicin - immunity protein complexes are amongst the most stable protein-protein complexes ever observed. The goal of this work is to gain understanding at the molecular level of the nature of these extremely tight protein-protein interactions. Furthermore, the crystal structure will provide information on the mechanism of inhibition by the immunity protein, as well as identification of colicin E3 residues likely involved in catalysis. Detailed knowledge of the enzyme-inhibitor interactions will al so shed light on the local structure of the ribosome in the vicinity of the cutting site. ***