The assembled structure of a complete tripartite bacterial multidrug efflux pump

Abstract

Bacteria like Escherichia coli and Pseudomonas aeruginosa expel drugs via tripartite multidrug efflux pumps spanning both inner and outer membranes and the intervening periplasm. In these pumps a periplasmic adaptor protein connects a substrate-binding inner membrane transporter to an outer membrane-anchored TolC-type exit duct. High-resolution structures of all 3 components are available, but a pump model has been precluded by the incomplete adaptor structure, because of the apparent disorder of its N and C termini. We reveal that the adaptor termini assemble a ␤-roll structure forming the final domain adjacent to the inner membrane. The completed structure enabled in vivo cross-linking to map intermolecular contacts between the adaptor AcrA and the transporter AcrB, defining a periplasmic interface between several transporter subdomains and the contiguous ␤-roll, ␤-barrel, and lipoyl domains of the adaptor. With short and long cross-links expressed as distance restraints, the flexible linear topology of the adaptor allowed a multidomain docking approach to model the transporter-adaptor complex, revealing that the adaptor docks to a transporter region of comparative stability distinct from those key to the proposed rotatory pump mechanism, putative drugbinding pockets, and the binding site of inhibitory DARPins. Finally, we combined this docking with our previous resolution of the AcrA hairpin-TolC interaction to develop a model of the assembled tripartite complex, satisfying all of the experimentally-derived distance constraints. This AcrA 3-AcrB3-TolC3 model presents a 610,000-Da, 270-Å-long efflux pump crossing the entire bacterial cell envelope.antibiotic resistance ͉ docked model ͉ membrane complex ͉ TolC exit duct

Amino Acid Substitution in the Major Multidrug Efflux Transporter Protein AcrB Contributes to Low Susceptibility to Azithromycin in Haemophilus influenzae

“…Although the mechanism by which this substitution confers nonsusceptibility to AZM remains unclear, the results of the EB efflux assay showed that AZM addition competed with EB efflux at the early stage, suggesting that this amino acid substitution contributed to alterations in initial recognition and efflux. AcrB is known to form a trimer that acts as a complex with the outer membrane channel TolC and the membrane fusion protein AcrA (10)(11)(12). This efflux system discharges the agents from both the inner membrane and the periplasm (10).…”

Structures of sequential open states in a symmetrical opening transition of the TolC exit duct

“…1, crosssection 3) interact with the apex of AcrB (9). On disruption of the bond network, in both TolC RS and TolC YFRS , the tips undergo immediate large changes (Fig.…”

“…The structures of all three drug efflux pump components are known: the trimeric TolC exit duct (5), the trimeric AcrB transporter (6,7), the partial adaptor AcrA (8), and the complete homologous Pseudomonas adaptor MexA (9,10). We have established a model of the assembled tripartite machinery on the basis of extensive site-specific cross-linking between the flexible, linearly arranged multidomain adaptor and its two cognate partner proteins (9,11). To recruit TolC, the 47 Å long α-hairpin domain of the adaptor packs against intramolecular grooves formed by entrance helices of each TolC protomer (9,11).…”

“…We have established a model of the assembled tripartite machinery on the basis of extensive site-specific cross-linking between the flexible, linearly arranged multidomain adaptor and its two cognate partner proteins (9,11). To recruit TolC, the 47 Å long α-hairpin domain of the adaptor packs against intramolecular grooves formed by entrance helices of each TolC protomer (9,11). Several domains of the adaptor bind to the IM transporter AcrB, while the TolC entrance helices make tip-to-tip contacts with AcrB (12, 13).…”

“…Several domains of the adaptor bind to the IM transporter AcrB, while the TolC entrance helices make tip-to-tip contacts with AcrB (12, 13). The assembly is stabilized by a single adaptor on each subunit, sealing the assembled pump against the periplasm (9).…”