Hierarchical order of critical residues on the immunity-determining regionof the lm7 protein which confer specific immunity to its cognate colicin

The directed mutagenesis study of the Im7 protein of colicin E7 revealed th at three residues, D31, D35, and E39, located in the loop 1 and helix 2 reg ions of the protein were critical for initiating the complex formation with its cognate colicin E7, Interestingly, the importance of these three criti cal residues in conferring specific immunity to its own colicin was exhibit ed in a hierarchical order, respectively. Moreover, we found that existence of the three critical residues was common among the DNase-type Im proteins . Most likely the three residues of the DNase-type immunity proteins are cr itical for initiating the unique protein-protein interactions with their co gnate colicin. In addition, replacement of the helix 2 of Im7 by the corres ponding region of Im8 produced a phenotype of the mutant protein very simil ar to that of Im8. This result suggests that the DNase-type Im proteins ind eed share a "homologous-structural framework" and evolution of the Im prote ins may be engendered by minor amino acid changes in this specific immunity -determining region without causing structural alteration of the proteins. (C) 1999 Academic Press.