PROTEIN-PROTEIN INTERACTIONS IN COLICIN E9 DNASE-IMMUNITY PROTEIN COMPLEXES .2. COGNATE AND NONCOGNATE INTERACTIONS THAT SPAN THE MILLIMOLAR TO FEMTOMOLAR AFFINITY RANGE

The in vivo and in vitro cross-binding of the colicin endonuclease-specific immunity proteins toward the DNase domain of colicin E9 is described. In vivo cross-protection was tested by toxin plate assays in which bacterial cells overexpressing each immunity (Im2, Im7, Im8, and Im9) were challenged with the Co1E9 toxin. Im9, the cognate immunity protein, renders cells completely resistant toward very high concentrations of the toxin (> 1 mg/mL), whereas the noncognate immunities display a spectrum of weaker cross-reactivities (< 0.01 mg/mL). The order of biological protection in this assay was Im9 much greater than Im2 > Im8, with Im7 providing no colicin E9 resistance. in vitro binding between the immunity proteins and the E9 DNase was analyzed by determining the dissociation constants for E9 DNase-Im protein complexes at pH 7.0 in the presence of 200 mM salt and at 25 degrees C. Stopped-flow fluorescence experiments suggest that both Im2 and Im8 associate with the E9 DNase by a two-step mechanism, in which the rate constants for both the bimolecular association (k(1) = similar to 6 x 10(7) M(-1) s(-1)) and the subsequent conformational change (k(2) + k(-2) = 4-5 s(-1)) are very similar to Im9 binding under the same conditions. Fluorescence chase experiments defined the dissociation rate constants for Im2 and Im8. The estimated values are 10(6)- and 10(8)-fold, respectively, faster than the off-rate for the Im9 protein. The ratio of the association and dissociation rate constants gives K-d values of approximately 10(-8) and 10(-6) M for Im2 and Im8, respectively, whereas the K-d for Im9 under these conditions is 10(-14) M. The activity of the E9 DNase was also analyzed by an in vitro plasmid nicking assay. All three noncognate immunity proteins inhibit the E9 DNase. The K-i's for Im2 and Im8 agree closely with the stopped-flow experiments, and the K-i for Im7 was determined to be 10(-4) M. The order of immunity protein affinity for the E9 DNase (Im9 much greater than Im2 > Im8 > Im7) is the same as that for the in vivo toxin protection experiments, implying a correlation between biological specificity and in vitro binding. Our results show that the specificity of E9 DNase-Im protein interactions are spread over an affinity range that spans more than 10 orders of magnitude and so offers new opportunities for understanding the basis for specificity in protein-protein interactions.