Eight reagents specifically modifying amino acids were applied to cells of a standard Escherichia coli colicin indicator strain to follow in vivo changes of its binding capacity for colicins E1-E3 and hence the binding domains (epitopes) for them in the outer membrane receptor protein BtuB. The effect of these reagents was also investigated in a mutant strain carrying an extensive BtuB deletion. The following differences of the binding epitopes could be ascertained. Colicin E1: Blockage of OH-groups, just as N-substitution of His and modification of Arg and Trp enhance binding of colicin E1. In the deleted receptor, also abolition of carboxylic anion bonds enhances its affinity for colicin E1. It follows that colicin E1 is bound, most of all, to the hydrophobic domain A (loops 1 + 2) of BtuB. Colicins E2 and E3: both exert rather analogous binding parameters. In contrast to E1, O-substitution of Ser and Thr dramatically decreases the E2 and E3 binding, similarly to modification of Lys. There is also a clear difference in the binding affinity of the domain for E2 and/or E3 and for E1 following modifications of their Arg and His. Colicins E2 and E3 are rather bound to the hydrophilic domain B (loops 5-7) of the receptor. In this respect, interactions of colicins E2 and E3 with deeper parts of A and B domains (Trp, several Arg, Lys and His residues) exhibited subtle differences. Acidic pH (4.5-6.0) shows a positive, while pH 7.0-8.5 a rather negative impact on the receptor-binding function for the colicins. It was clearly demonstrated that there is just a partial difference between the binding behavior of colicins E1, E2 and/or E3.

Department of Biology Faculty of Medicine Masaryk University 662 44 Brno Czechia

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