Crystal Structure of Colicin E3

9728420 Shoham本研究的目标是确定结肠素E3与其抑制物免疫蛋白的复合体的晶体结构。已经找到了三个合适的重原子衍生物，并将寻找更多。不对称单元中的四个分子对应的电子密度将被平均，以提高MAP的可解释性。在结构测定完成后，将设计结肠素E3和免疫蛋白的定点突变体，以探索某些残基对亲和力的贡献。这些突变体将在体外和体内从结构和功能上进行表征。Colicin E3是一种有毒蛋白质，由某些E.Coli菌株分泌，以消除生活在同一生态位中的相关菌株。Colicin E3执行三个功能：它与靶细胞包膜上的特定受体结合，穿过敏感细胞内膜内化到敏感细胞中，一旦进入细胞，它就会通过划破16S核糖体RNA上的特定键来灭活感染细胞的蛋白质生物合成机制。因此，粘菌素E3的毒性源于其酶活性。这三种功能明显定位于结肠素E3分子的三个不同结构域上。由于一种“免疫蛋白”与粘菌素E3以1：1的复合体紧密结合，从而使其失去活性，因此产生的生物体对粘菌素E3的毒性具有免疫力。Colicin-免疫蛋白质复合体是迄今观察到的最稳定的蛋白质-蛋白质复合体之一。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，晶体结构将提供免疫蛋白抑制机制的信息，以及鉴定可能参与催化的粘菌素E3残基。对酶-抑制物相互作用的详细了解也将有助于了解切割部位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. ***