Chunnel vision. Export and efflux through bacterial channel-tunnels

Abstract

The Escherichia coli TolC protein is central to toxin export and drug efflux across the inner and outer cell membranes and the intervening periplasmic space. The crystal structure has revealed that TolC assembles into a remarkable alpha-helical trans-periplasmic cylinder (tunnel) embedded in the outer membrane by a contiguous beta-barrel (channel), so providing a large duct open to the outside environment. The channel-tunnel structure is conserved in TolC homologues throughout Gram-negative bacteria, and it is envisaged that they are recruited and opened, through a common mechanism, by substrate-specific inner-membrane complexes.

Fig. 1. Phylogenetic tree of the TolC family. The 36 TolC homologues for which function is known, or strongly implicated by the location of their genes in the export or efflux operons, sorted by TREEVIEW (Page, 1996) on the basis of a sequence alignment using MULTIALIN (Corpet, 1988). Abbreviations: Ea, Erwinia amylovora; Ec, Escherichia coli; Ech, Erwinia chrysanthemi; Se, Salmonella enteritidis; Sm, Serratia marcescens; Pa, Pseudomonas aeruginosa; Pf, Pseudomonas fluorescens. SilC¹ may be an exception to the subgrouping as it is reported to be involved in silver efflux. Scale ‘0.1’ indicates 0.1 nucleotide substitutions. Note: the database has a further 20 homologues, with no ascribed function to date.

Fig. 2. The structures of bacterial OM proteins. (i) Top view of the proteins down the OM channel. In the case of TolC this extends to ∼2/3 of the height, i.e. the top 100 Å. (ii) Side view, at right angles to the plane of the OM. (iii) Cross-section of TolC near the tunnel entrance, i.e. the bottom 40 Å. Red, green and blue indicate individual monomers.

Fig. 3. Sequence conservation of TolC homologues related to the TolC structure. The TolC monomer coloured, as indicated, according to the level of consensus of individual amino acids throughout the known 56 TolC homologues. Alignment was performed using MULTIALIN (Corpet, 1988), consensus scores were calculated using the CLUSTAL function with matrix PAM350.

Fig. 4. Channel-tunnels of the TolC family acting in export and efflux. A model indicating reversible interaction of trimeric TolC homologues (green) with substrate-specific IM complexes containing an adaptor protein (red/blue) and an energy-providing protein of either the traffic ATPase (protein export, yellow) or proton antiporter families (small molecule efflux, purple). Escherichia coli TolC can act in both pathways, while in other bacteria multiple TolC homologues act separately in distinct pathways. In response to substrate engagement [a peptide chain, or hydrophilic and hydrophobic drugs (stars)], the trimeric adaptor protein contacts the periplasmic tunnel, possibly via the predicted coiled-coil structures indicated, prompting the conformational change that opens the entrance.