Discovery of critical Tol A-binding residues in the bactericidal toxin colicin N: a biophysical approach.
Colicins translocate across the Escherichia coli outer membrane and periplasm by interacting with several receptors. After first binding to outer membrane surface receptors via their central region, they interact with TolA or TonB proteins via their N-terminal regions. Finally, the toxic C-terminal region is inserted into or across the cytoplasmic membrane. We have measured the binding of colicin N to TolA by isothermal titration microcalorimetry (ITC) and tryptophan fluorescence. The isolated N-terminal domain exhibits a higher affinity for TolA (Kd = 1 microM) than does the whole colicin (18 microM), and similar behaviour has been observed when the N-terminal domain of the g3p protein of the bacteriophage fd, which also binds TolA, is examined in isolation and in situ. This may indicate a similar mechanism in which a cryptic TolA binding site is revealed after primary receptor binding. The isolated colicin N N-terminal domain appears to be unstructured in circular dichroism and fluorescence studies. We have used mutagenesis and ITC to characterize the TolA binding site and have shown it to be of a different sequence and much further from the N-terminus than previously thought.
Department of Biochemistry and Genetics, The Medical School, University of Newcastle Upon Tyne, UK.
E M
EM
Raggett
G
G
Bainbridge
L J
LJ
Evans
A
A
Cooper
J H
JH
Lakey
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D002151:Calorimetry; D002942:Circular Dichroism; D003087:Colicins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005453:Fluorescence; D008969:Molecular Sequence Data; D016296:Mutagenesis; D018272:Porins / Q000378:metabolism; D013816:Thermodynamics; D014364:Tryptophan / Q000032:analysis
D001426:Bacterial Proteins; D003087:Colicins; D029968:Escherichia coli Proteins; C109237:OmpF protein; D018272:Porins; C062093:tolA protein, E coli; D014364:Tryptophan