The central domain of colicin N possesses the receptor recognition site but not the binding affinity of the whole toxin.
Colicin N is a three-domain pore-forming colicin which kills enterobacterial cells following an initial binding to its receptor, the outer membrane porin OmpF. The receptor-binding domain of colicin N alone, and attached to the translocation domain, was overexpressed and purified using a hexahistidine tag. The receptor domain attached to the pore-forming domain was obtained by enzymatic digestion. Circular dichroism spectroscopy showed that the domains have structure in keeping with the known structure of colicin N. The receptor domain was stable, retaining both secondary and tertiary structure in 2 M guanidine hydrochloride and at low pH. It bound to both OmpF and PhoE porin-producing Escherichia coli with no toxicity and protected sensitive E. coli against intact colicin N toxicity at high domain/ colicin N ratios. Its in vitro affinity for OmpF, as determined by isothermal titration microcalorimetry, was found to be approximately 50-fold weaker than that of native colicin N. The receptor domain was readily out-competed by native colicin N in in vivo fluorescence assays which, coupled with its structural stability, suggests that its interaction with OmpF is one of weak, reversible binding. Since neither of the double domain constructs shows wild-type binding affinity either, it appears that the molecular recognition is a property of the receptor domain but that affinity is influenced by the entire molecule.
Evans
L J
LJ
Department of Biochemistry and Genetics, Medical School, University of Newcastle Upon Tyne, United Kingdom.
Labeit
S
S
Cooper
A
A
Bond
L H
LH
Lakey
J H
JH
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002151:Calorimetry; D002942:Circular Dichroism; D003087:Colicins / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D001425:Bacterial Outer Membrane Proteins; D003087:Colicins; D029968:Escherichia coli Proteins; D011956:Receptors, Cell Surface; C028593:colicin receptor, E coli