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TolA is an inner membrane protein with three domains: a transmembrane N‐terminus and periplasmic central and C‐terminal domains. The interaction of TolA with outer membrane porins of Escherichia coli was investigated. Western blot analyses of cell extracts with anti‐TolA antibodies indicated that TolA forms high molecular weight complexes specifically with trimeric OmpF, OmpC, PhoE and LamB, but not with OmpA. The interaction of purified TolA domains with purified porins was also studied. TolA interacted with OmpF, PhoE and LamB porins via its central domain, but not with either their denatured monomeric forms or OmpA. Moreover, the presence or absence of lipopolysaccharides associated with trimeric porins did not modify the interactions. These results suggest that the specific interaction of TolA with outer membrane porins might be relevant to the function of Tol proteins.

Colicins are divided into two groups according t o the proteins required for their import into sensitive bacteria. The To1 and TonB pathways are involved in import of group A and group B colicins respectively. Because previous analyses have shown that colicin E l and colicin A (two group A colicins) interact in witro 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 threcdomain protein with an N-terminal innermembrane anchor and a long periplasmic region formed by two domains (TolAII and TolAlll). TolAIII, TolAll and TolAII-Ill soluble domains with an Nterminal 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 t o TolAlll were estimated by surface plasmon resonance and were shown t o be similar.

ABSTRACT:Colicins are killer proteins that use envelope proteins from the outer and the inner membranes to reach their cellular target in susceptible cells of Escherichia coli. Each group A colicin uses a combination of Tol proteins to cross the outer membrane of gram-negative bacteria and to exert their killing activity. The TolA protein, necessary for the import of all the group A colicins, is a 421-amino acid residue protein composed of three domains (TolAI, TolAII, and TolAIII). TolAIII interacts with the N-terminal domain of colicin A (AT1). Analytical ultracentrifugation reveals that TolAII and TolAIII are monomer structures, TolAII has an elongated structure, and TolAIII is rather globular. Circular dichroism (CD) spectra were done with TolAII-III, TolAII, TolAIII, AT1, and the AT1-TolAII-III complex. TolA CD spectra reveal the presence of ␣-helix structure in aqueous solution and the intensity of the ␣-helix signal is the highest with TolAII. Few structural changes are observed with the complex AT1-TolAII-III. Molecular modeling was done for TolAII-III, taking into account CD and ultracentrifugation data and show that domain II can adopt a barrel structure made of three twisted ␣-helices similar to coiled coil helices while domain III can adopt a globular structure.