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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 (k1) 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 Kd for the complex (k(off)/k1) 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.(ABSTRACT TRUNCATED AT 250 WORDS)

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Binding of enzymatic E colicins to the vitamin B12 receptor, BtuB, is the first stage in a cascade of events that culminate in the translocation of the cytotoxic nuclease into the Escherichia coli cytoplasm and release of its tightly bound immunity protein. A dogma of colicin biology is that the toxin coiled-coil connecting its functional domains must unfold or unfurl to span the periplasm, with recent reports claiming this reaction is initiated by receptor binding. We report isothermal titration calorimetry data of BtuB binding the endonuclease toxin ColE9 and a disulfide form (ColE9 S-S ) where unfolding of the coiled-coil is prevented and, as a consequence, the toxin is biologically inactive. Contrary to expectation, the thermodynamics of receptor binding, characterized by large negative values for T⌬S, are identical for the two colicins, arguing against any form of BtuB-induced unfolding. We go on to delineate key features of the ''colicin translocon'' that assembles at the cell surface after BtuB binding by using a complex of histidinetagged Im9 bound to ColE9 S-S . First, we show that the porin OmpF is recruited directly to the BtuB⅐colicin complex to form the translocon. Second, recruitment is through the natively unfolded region of the colicin translocation domain, with this domain likely having two contact points for OmpF. Finally, the immunity protein is not released during its assembly. Our study demonstrates that although colicin unfolding is undoubtedly a prerequisite for E. coli cell death, it must occur after assembly of the translocon.colicin ͉ toxin ͉ outer membrane ͉ translocation ͉ native disorder