The Crystal Structure of the Outer Membrane Protein VceC from the Bacterial Pathogen Vibrio cholerae at 1.8 Å Resolution

Abstract

Multidrug resistance in Gram-negative bacteria arises in part from the activities of tripartite drug efflux pumps. In the pathogen Vibrio cholerae, one such pump comprises the inner membrane proton antiporter VceB, the periplasmic adaptor VceA, and the outer membrane channel VceC. Here, we report the crystal structure of VceC at 1.8 Å resolution. The trimeric VceC is organized in the crystal lattice within laminar arrays that resemble membranes. A well resolved detergent molecule within this array interacts with the transmembrane ␤-barrel domain in a fashion that may mimic proteinlipopolysaccharide contacts. Our analyses of the external surfaces of VceC and other channel proteins suggest that different classes of efflux pumps have distinct architectures. We discuss the implications of these findings for mechanisms of drug and protein export.To expel drugs and other toxic compounds, Gram-negative bacteria use specialized machinery that guides the compounds across two membranes and over the separating interstitial space, known as the periplasm. One type of such transport machinery is the complex formed by an inner membrane proton antiporter, an outer membrane channel, and a periplasmic protein that consolidates the assembly (1, 2). These energy-dependent tripartite pumps extrude actively a variety of noxious compounds from the cytoplasm or inner membrane to the extracellular medium. A number of such pumps have been implicated in multidrug resistance of Gram-negative species, and representative examples that have been well characterized include the Escherichia coli AcrAB-TolC and the Pseudomonas aeruginosa MexAB-OprM assemblies (3, 4).Advances are being made in understanding the mechanism of multidrug efflux in Gram-negative bacteria at the level of stereochemistry. To date, high resolution crystal structures have become available for the outer membrane components TolC (5) and OprM (6), the inner membrane proton antiporter AcrB (7,8), and the periplasmic adaptor MexA (9, 10). Based on these structures, several models have been proposed for the organization of the AcrAB-TolC assembly as a representative efflux pump (9 -11). However, the detailed interactions of the components have not been established nor is it clear how the transport of drugs is coupled to proton translocation.Tripartite pumps are likely to contribute to drug resistance of the Gram-negative pathogen Vibrio cholerae, which is the causative agent of the disease cholera. Isolates of V. cholerae have been described that are resistant to chemically diverse antibiotics, such as ampicillin, penicillin, streptomycin, nitrofurantoin, and erythromycin, as well as to toxic metals like Pb 2ϩ and Zn 2ϩ (12). A putative tripartite pump has been identified from V. cholerae comprising the inner membrane antiporter VceB, the periplasmic adaptor protein VceA, and the outer membrane channel VceC (13). Although the function of this pump in V. cholerea has not been demonstrated, its components were found to collectively complement the multidrug resistance phenotype in ...

TdeA, a TolC-like protein required for toxin and drug export in Aggregatibacter (Actinobacillus) actinomycetemcomitans

“…Indeed, Andersen et al (Andersen et al, 2000) noted that TolC-like proteins share remarkable structural similarities with each other, but often lack significant sequence similarities. Structural predictions for TdeA correspond closely with known structures of other bacterial TolC proteins including the presence of a leader peptide, the number and locations of β -sheets (which form the outer membrane pore) and α -helical domains, and internal similarity between the two halves of the protein Koronakis et al, 2004;Federici et al, 2005).…”

“…The closest related proteins for which biological activity has been demonstrated and crystal structures are available are VceC from Vibiro cholerae (21% amino acid identity) (Federici et al, 2005) and E. coli TolC (21% amino acid identity) Koronakis, 2003).…”

A periplasmic coiled-coil interface underlying TolC recruitment and the assembly of bacterial drug efflux pumps

“…In this solution the adaptor contacts the intramolecular groove between the inner and outer entrance coils of TolC H7/H8/H3 (Fig. 3B), in contrast to computational models that suggested the adaptor contacts the groove at the TolC subunit interface formed by helices H3/H4 and H7* of the adjacent monomer (21,26,27). In the simplest interpretation, our results suggest a single binding site on the TolC protomer, interacting with the ␣-helical hairpin of a single adapter molecule.…”

“…Tight conformational restraints were applied to produce rigid body docking, except in the equatorial region of TolC where restraints were relaxed (residues 408, 421, and 195-207) to allow flexibility. This is a legitimate approach because, though ordered in the TolC crystal structure, the equatorial domain is conformationally variable or even disordered in other crystallized examples of the TolC family (27).…”

“…Hypothetical models of pump assembly reflect this (21,26,27), but there has been no substantive experimental study of TolC-adaptor intermolecular contacts essential to this key recruitment interaction. We have carried out systematic in vivo site-specific cross-linking to arrays of cysteine residues introduced into the periplasmic domains of TolC and separately into the ␣-helical hairpin of AcrA.…”