The TolA-recognition site of colicin N. ITC, SPR and stopped-flow fluorescence define a crucial 27-residue segment.
Colicins translocate across the Escherichia coli outer membrane and periplasm by interacting with several receptors. After first binding to the outer membrane surface receptors via their central region, they interact with TolA or TonB proteins via their N-terminal region. Colicin N residues critical to TolA binding have been discovered, but the full extent of any colicin TolA site is unknown. We present, for the first time, a fully mapped TolA binding site for a colicin. It was determined through the use of alanine-scanning mutants, glutathione S-transferase fusion peptides and Biacore/fluorescence binding studies. The minimal TolA binding region is 27 residues and of similar size to the TolA binding region of bacteriophage g3p-D1 protein. Stopped-flow kinetic studies show that the binding to TolA follows slow association kinetics. The role of other E. coli Tol proteins in colicin translocation was also investigated. Isothermal titration microcalorimetry (ITC) and in vivo studies conclusively show that colicin N translocation does not require the presence of TolB. ITC also demonstrated colicin A interaction with TolB, and that colicin A in its native state does not interact with TolAII-III. Colicin N does not bind TolR-II. The TolA protein is shown to be unsuitable for direct immobilisation in Biacore analysis.
Gokce
I
I
Department of Chemistry Faculty of Science, Gaziomanpasa University, Tokat, Turkey.
Raggett
E M
EM
Hong
Q
Q
Virden
R
R
Cooper
A
A
Lakey
J H
JH
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D002151:Calorimetry; D002942:Circular Dichroism; D003087:Colicins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D005453:Fluorescence; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D033903:Periplasmic Proteins; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance; D017186:Titrimetry; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D003087:Colicins; D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D011993:Recombinant Fusion Proteins; C062093:tolA protein, E coli; C062094:tolB protein, E coli; D014364:Tryptophan