Structure of the AcrABZ-TolC Multidrug Efflux Pump in a Drug-Bound State

Abstract

The capacity of numerous bacterial species to tolerate antibiotics and other toxic compounds arises in part from the activity of energy-dependent transporters. In Gram-negative species, the transporters are components of nanomachines that span the cell envelope, and a well characterized representative of such assemblies is the AcrABZ-TolC complex, a acridine resistance complex pump of Escherichia coli. The AcrABZ-TolC assembly comprises the outer-membrane channel TolC, the secondary transporter AcrB located in the inner membrane, the periplasmic AcrA which bridges these two integral membrane proteins, and a 49 amino acid residue protein, AcrZ. The entire AcrABZ-TolC complex has a molecular mass of ∼850 kDa. In E. coli, the AcrABZ-TolC pump transports vectorially chemically similar compounds thereby conferring bacterial resistance to a broad spectrum of antibiotics.In our previous study, we have determined a substrate-free structure of the entire AcrABZ-TolC pump at 16 A resolution using single-particle cryo-EM approach (Du, Wang, et al Nature 509, 512-5, 2014). We were able to build a pseudo-atomic model of the pump utilizing available X-ray crystal structures of its components. This model defines the quaternary arrangement of the pump components and identifies key domain interaction interfaces critical for the channel assembly and opening.Recently, using single-particle cryo-EM we have resolved the structure of AcrABZ-TolC pump bound to puromycin to subnanometer resolution. The cryo-EM density map resolves the secondary structural elements of the components and clearly identifies molecular boundaries between individual components of the pump. The new structure provides insights into conformational changes that might be involved in substrate binding and transport. Further studies are being pursued to decipher the molecular mechanisms underlying assembly and function of the AcrABZ-TolC pump.