Structural Studies on the Translocation of Colicin E3 into Sensitive Cells

Colicin E3是一种有毒蛋白质，由某些菌株分泌，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：它与靶细胞膜上的BtuB受体结合，穿过细胞膜内化到敏感细胞，一旦进入细胞，它通过在核糖体A位点A1493和G1494之间的16S核糖体RNA缺口来灭活感染细胞的蛋白质生物合成机制。产生的生物体通过一种“免疫蛋白”(IP)对Colicin E3的毒性免疫，该免疫蛋白以1：1的复合体与Colicin E3紧密结合，从而使其失去活性。该项目的目标是了解结肠素E3与促进其跨大肠杆菌外膜和内膜转运的蛋白质的相互作用。本研究将集中于三个三元络合物的晶体结构测定：(I)Colicin E3，IP和TolA的C-末端片段；(Ii)Colicin E3，IP和TolB；(Iii)Colicin E3，IP和BtuB细胞表面受体。这项工作的另一部分涉及到结肠素E3结构本身以及在IP、Tol蛋白或BtuB受体存在的情况下的动态方面。灵活性的程度，无论是分段的还是全局的，将通过荧光能量转移(FRET)来衡量。Colicin E3-IP二元复合体的晶体结构表明，在没有IP的情况下，在结构域结点处形成了柔性铰链。这将通过FRET实验进行测试。这些研究将有助于阐明这种多功能蛋白质的作用机制。

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.