Molecular analysis of the protein‐protein interaction between the E9 immunity protein and colicin E9

Abstract

The specificity‐determining region of the colicin E9 immunity protein (Im9) for its interaction with its cognate E colicin has been localized to residues 16–43 of the 86‐amino‐acid protein by the use of gene fusions. A comparison of the alignment of residues in this region of the Im2, Im8 and Im9 proteins have identified nine candidate specificity‐determining residues. Using site‐directed mutagenesis, we have changed each of these residues in the Im9 protein to the residue found in the same position in the Im8 protein. The immunity phenotype conferred by the mutant immunity protein was then tested. Of the nine residues, only one (Val34 to Asp) showed any evidence of conferring immunity to colicin E8. Changing other residues in the specificity‐determining region to the equivalent Im8 residue did not affect the phenotype conferred by the mutant protein, with the exception of the change of Val37 to Glu, which resulted in low‐level E8 immunity. While the substitutions at positions 34 and 37 of the Im9 protein introduced immunity towards ColE8, they did not diminish the immunity towards ColE9, suggesting that the two immunity proteins may have a common specificity framework which can be modified by single mutations. In addition, we have used chemical modification of the unique cysteine residue of Im9 (Cys23) in order to probe further this specificity‐determining region. Cys23 in the purified Im9 protein is accessible to modification with the thiol‐specific reagent 5,5′‐dithiobis(2‐nitrobenzoic acid) and the stoichiometry of labelling is close to 1:1. This residue, however, cannot be labelled by 5,5′‐dithiobis(2‐nitrobenzoic acid) when the Im9 protein is complexed to colicin E9. This result is consistent with the Cys23 residue being buried in the complex. However, when the purified Im9 protein modified at Cys23 with a variety of reagents was used in DNase inhibition assays with colicin E9, the modified Im9 proteins still possessed anti‐DNase activity but only up to a certain derivative molecular mass. These results are discussed in terms of the proximity of Cys23 to the specificity‐determining region. Copyright © 1992, Wiley Blackwell. All rights reserved