PROTEIN-PROTEIN INTERACTIONS IN COLICIN E9 DNASE-IMMUNITY PROTEIN COMPLEXES .1. DIFFUSION-CONTROLLED ASSOCIATION AND FEMTOMOLAR BINDING FOR THE COGNATE COMPLEX

The cytotoxic activity of the secreted bacterial toxin colicin E9 is due to a nonspecific DNase housed in the C-terminus of the protein. A kinetic and thermodynamic analysis of complex formation for both the holotoxin and the isolated DNase domain with the cytoplasmic inhibitor of this enzyme, the immunity protein Im9, is presented. The dissociation constant for each complex was calculated from the ratio of the association and dissociation rate constants. Association was monitored by stopped-flow fluorescence and comprises at least two steps for both complexes, an initial fluorescence enhancement followed by a fluorescence quench. The data are consistent with a two-step binding mechanism in which the rate of formation of an encounter complex (k(1)) is rate determining and essentially diffusion controlled (4.0 x 10(9) M(-1) s(-1) for colicin E9) in buffer of low ionic strength. This encounter complex then rearranges to the final stable complex. Sequential stopped-flow experiments using 5-hydroxy-L-tryptophan labeled DNase domain support the two-step mechanism and further show that the rate of encounter complex rearrangement is significantly faster than its dissociation. The overall rate of dissociation of the colicin E9-Im9 complex (k(off)) was determined by radioactive subunit exchange to be 3.7 x 10(-7) s(-1). Thus, the K-d for the complex (k(off)/k(1)) is 9.3 x 10(-17) M, which corresponds to a change in free energy on binding of -21.9 kcal mol(-1) at 25 degrees C. The affinity of the complex between the isolated DNase domain of colicin E9 and Im9 was very similar to that of the full size protein (K-d = 7.2 x 10(-17) M, Delta G = -22 kcal mol(-1)). The effects of pH, temperature, and salt on complex formation were also analyzed. Altering the pH of the medium (between pH 5 and 9) had little effect on the association and dissociation rate constants. The temperature dependence of the equilibrium constant yielded values for Delta H of -6.4 kcal mol(-1) and for Delta S of 52.3 cal mol(-1) K-1 at 25 degrees C. The association rate constant decreases by over two orders of magnitude in the presence of 250 mM NaCl, suggesting that electrostatic steering plays an important role in the rapid formation of the encounter complex.