Binding of colicins A and E1 to purified ToIA domains

Abstract

Colicins are divided into two groups according to the proteins required for their import into sensitive bacteria. The Tol and TonB pathways are involved in import of group A and group B colicins respectively. Because previous analyses have shown that colicin El and colicin A (two group A colicins) interact in vitro with the C-terminal domain of TolA (TolAlll) while colicin B (group B colicin)does not, attention was focused on these interactions with purified proteins.TolA has been described as a three-domain protein with an N-terminal inner-membrane anchor and a long periplasmic region formed by two domains(TolAII and TolAlll). TolAIII, TolAll and TolAII-Ill soluble domains with an N-terminal hexa-histidine extension were purified. The interactions of colicins with the purified TolA domains were analysed by overlay Western blotting,which indicated that both N-terminal domains of colicins A and E l interacted with TolAIII, while a gel shift procedure detected no interaction with colicin El.The binding kinetic values of the N-terminal domains of colicins A and E l to TolAlll were estimated by surface plasmon resonance and were shown to be similar.