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. ***
