Macromolecular import into Escherichia coli:: The TolA C-terminal domain changes conformation when interacting with the colicin A toxin

Various macromolecules such as bacteriotoxins and phage DNA parasitize some envelope proteins of Escherichia coli to infect the bacteria. A two-step import mechanism involves the primary interaction with an outer membrane receptor or with a pilus followed by the translocation across the outer membrane. However, this second step is poorly understood. It was shown that the To1A, To1Q, and To1R proteins play a critical role in the translocation of group A colicins and filamentous bacteriophage minor coat proteins (g3p). Translocation of these proteins requires the interaction of their N-terminal domain with the C-terminal domain of To1A (To1AIII). In this work, short soluble To1AIII domains were overproduced in the cytoplasm and in the periplasm of E. coli. In To1AIII, the two cysteine residues were found to be reduced in the cytoplasmic form and oxidized in the periplasmic form. The interaction of To1AIII with the N-terminal domain of colicin A (ATh) is observed in the presence and in the absence of the disulfide bridge. The complex formation of To1AIII and ATh was found to be independent of the ionic strength. An NMR study of To1AIII, both free and bound, shows a significant structural change when interacting with ATh, in the presence or absence of the disulfide bridge. In contrast, such a structural modification was not observed when To1AIII interacts with gap N1. These results suggest that bacteriotoxins and Ff bacteriophages parasitize E. coli using different interactions between To1A and the translocation domain of the colicin and gap protein, respectively.