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 ColE9 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 >> 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 (k1 = approximately 6 x 10(7) M-1 s-1) and the subsequent conformational change (k2 + 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Wallis
R
R
School of Biological Sciences, University of East Anglia, Norwich, U.K.
Leung
K Y
KY
Pommer
A J
AJ
Videler
H
H
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*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D005453:Fluorescence; D007700:Kinetics; D011485:Protein Binding; D013816:Thermodynamics
D001426:Bacterial Proteins; D003087:Colicins; D029968:Escherichia coli Proteins; C007104:colicin immunity proteins; C488859:immE9 protein, E coli; D003851:Deoxyribonucleases