Abstract
The pathway of cellular entry of colicins and viral DNA is a fundamental structure problem that is relevant to an understanding the molecular basis of infectious diseases. The cytotoxin colicin E1 uses the outer membrane/trans-periplasmic drug export protein, TolC, for its import. TolC consists of a 12 strand OM β-barrel connected to a 12 strand α-helical tunnel that defines a pathway through the peptidoglycan barrier in the periplasm to the cytoplasmic membrane in which the C-terminal domain of colicin E1 inserts and forms a depolarizing ion channel. The nature of the interaction of the colicin with TolC and the mechanism of its translocation are unknown. Previous studies with planar bilayers showed that colicin E1 occludes TolC channels [1], as do certain colicin T-domain peptides [2].Here, in vivo protection of sensitive E. coli from colicin E1 by a series of N-terminal colicin peptides is used to probe the interaction of the colicin with TolC, with the goal of defining the sites of TolC-colicin interaction and the mechanism of colicin entry. N-terminal segments ‘1-40’,’1-81’, and ‘1-100’ of the colicin did not provide cytotoxic protection, nor occlude TolC channels. Segments ‘1-120’, ‘1-140’, 1-190’, as well as ‘41-190’ and ‘57-190’ protected efficiently in vivo and occluded TolC with high efficiency. Occlusion required a trans-negative electrical potential and was irreversible. Co-elution of the colicin peptides with TolC on a Superdex 200 column was shown for ‘41-190’, but not for ‘1-81.’ In addition to the correlation with protection in vivo from killing by colicin E1, occlusion efficiency also correlated with a basic pI between colicin residues 82 and 140. [1] Biophys. J., 87:3901-3911, 2004; [2] Biochem. Soc. Trans., 40:1463-1468, 2012. Support: NIH AI091633 (KSJ); NIH GM38323 and the Henry Koffler Professorship (WAC).
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