Structural Studies on the Translocation of Colicin E3 into Sensitive Cells

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细菌菌株。大肠杆菌素E3具有三种功能：它与靶细胞包膜上的BtuB受体结合，穿过细胞质膜内化到敏感细胞中，一旦进入细胞，它通过在核糖体A位点A1493和G1494之间切割16 S核糖体RNA来灭活感染细胞的蛋白质生物合成机制。生产生物体通过“免疫蛋白”（IP）对大肠杆菌素E3的毒性免疫，该免疫蛋白以1：1的复合物与大肠杆菌素E3紧密结合，从而使其失活。本项目的目标是了解大肠杆菌素E3与蛋白质之间的相互作用，这些蛋白质有助于大肠杆菌素E3穿过大肠杆菌的外膜和内膜。杆菌研究将集中在三个三元复合物的晶体结构测定：（i）大肠杆菌素E3，IP和托拉的C-末端片段;（ii）大肠杆菌素E3，IP和TolB;（iii）大肠杆菌素E3，IP和BtuB细胞表面受体。这项工作的另一部分涉及大肠杆菌素E3结构本身的动态方面，并在IP或Tol蛋白或BtuB受体的存在下。通过荧光能量转移（FRET）测量节段或整体的柔韧性程度。大肠杆菌素E3-IP二元复合物的晶体结构表明，在IP的情况下，在域连接处形成柔性铰链。这将通过FRET实验进行测试。这些研究将阐明这种多功能蛋白的作用机制。

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 the BtuB receptor on the target cell envelope, it is internalized into sensitive cells across their cytoplasmic membrane, and once inside the cell, it inactivates the protein biosynthetic machinery of the infected cell by nicking 16S ribosomal RNA between A1493 and G1494 at the ribosomal A site. The producing organism is immune to the toxicity of colicin E3 by virtue of an "immunity protein" (IP), which tightly binds to colicin E3 in a 1:1 complex, and thus renders it inactive. The goal of this project is to understand the interaction of colicin E3 with proteins that facilitate its translocation across the outer and inner membranes of E. coli. The research will focus on the crystal structure determination of three ternary complexes: (i) colicin E3, IP and a C-terminal fragment of TolA; (ii) colicin E3, IP and TolB; (iii) colicin E3, IP and the BtuB cell surface receptor. Another part of this work deals with the dynamic aspects of the colicin E3 structure by itself and in the presence of either IP or the Tol proteins or the BtuB receptor. The degree of flexibility, either segmental or global, will be measured by Fluorescence Energy Transfer (FRET). The crystal structure of the colicin E3-IP binary complex suggests the formation of flexible hinges at the domain junctions in the absence of IP. This will be tested by FRET experiments. These studies will shed light on the mechanism of action of this multifunctional protein.