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 peripla sm by interacting with several receptors. After first binding to the outer membrane surface receptors via their central region, they interact with Tol A 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 T olA site is unknown. We present, for the first time, a fully mapped TolA bi nding site for a colicin. It tvas determined through the use of alanine-sca nning mutants, glutathione S-transferase fusion peptides and Biacore/fluore scence 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 as sociation kinetics. The role of other E. coil Tol proteins in colicin trans location was also investigated. Isothermal titration microcalorimetry (ITC) and in vivo studies conclusively show that colicin N translocation does no t 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 T olAII-III. Colicin N does not bind TolR-II. The TolA protein is shown to be unsuitable for direct immobilisation in Biacore analysis. (C) 2000 Academi c Press.