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

The cytotoxic activity of the secreted bacterial toxin colicin E9 is d ue to a nonspecific DNase housed in the C-terminus of the protein. A k inetic and thermodynamic analysis of complex formation for both the ho lotoxin and the isolated DNase domain with the cytoplasmic inhibitor o f this enzyme, the immunity protein Im9, is presented. The dissociatio n constant for each complex was calculated from the ratio of the assoc iation and dissociation rate constants. Association was monitored by s topped-flow fluorescence and comprises at least two steps for both com plexes, 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 det ermining and essentially diffusion controlled (4.0 x 10(9) M(-1) s(-1) for colicin E9) in buffer of low ionic strength. This encounter compl ex then rearranges to the final stable complex. Sequential stopped-flo w experiments using 5-hydroxy-L-tryptophan labeled DNase domain suppor t the two-step mechanism and further show that the rate of encounter c omplex rearrangement is significantly faster than its dissociation. Th e 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, whi ch 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 fu ll size protein (K-d = 7.2 x 10(-17) M, Delta G = -22 kcal mol(-1)). T he effects of pH, temperature, and salt on complex formation were also analyzed. Altering the pH of the medium (between pH 5 and 9) had litt le effect on the association and dissociation rate constants. The temp erature dependence of the equilibrium constant yielded values for Delt a 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 electros tatic steering plays an important role in the rapid formation of the e ncounter complex.