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 (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)
Wallis
R
R
School of Biological Sciences, University of East Anglia, Norwich, U.K.
Moore
G R
GR
James
R
R
Kleanthous
C
C
D001426:Bacterial Proteins / Q000378:metabolism*; D003087:Colicins / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004058:Diffusion; D004791:Enzyme Inhibitors / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D005470:Fluorometry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D013816:Thermodynamics
D001426:Bacterial Proteins; D003087:Colicins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; C007104:colicin immunity proteins; C488859:immE9 protein, E coli; D012965:Sodium Chloride; D003851:Deoxyribonucleases