Coupling of remote alternating-access transport mechanisms for protons and substrates in the multidrug efflux pump AcrB.

Membrane transporters of the RND superfamily confer multidrug resistance to pathogenic bacteria, and are essential for cholesterol metabolism and embryonic development in humans. We use high-resolution X-ray crystallography and computational methods to delineate the mechanism of the homotrimeric RND-type proton/drug antiporter AcrB, the active component of the major efflux system AcrAB-TolC in Escherichia coli, and one most complex and intriguing membrane transporters known to date. Analysis of wildtype AcrB and four functionally-inactive variants reveals an unprecedented mechanism that involves two remote alternating-access conformational cycles within each protomer, namely one for protons in the transmembrane region and another for drugs in the periplasmic domain, 50 Å apart. Each of these cycles entails two distinct types of collective motions of two structural repeats, coupled by flanking α-helices that project from the membrane. Moreover, we rationalize how the cross-talk among protomers across the trimerization interface might lead to a more kinetically efficient efflux system.

Transport of drugs by the multidrug transporter AcrB involves an access and a deep binding pocket that are separated by a switch-loop.

AcrAB-TolC is the major efflux protein complex in Escherichia coli extruding a vast variety of antimicrobial agents from the cell. The inner membrane component AcrB is a homotrimer, and it has been postulated that the monomers cycle consecutively through three conformational stages designated loose (L), tight (T), and open (O) in a concerted fashion. Binding of drugs has been shown at a periplasmic deep binding pocket in the T conformation. The initial drug-binding step and transport toward this drug-binding site has been elusive thus far. Here we report high resolution structures (1.9-2.25 Å) of AcrB/designed ankyrin repeat protein (DARPin) complexes with bound minocycline or doxorubicin. In the AcrB/doxorubicin cocrystal structure, binding of three doxorubicin molecules is apparent, with one doxorubicin molecule bound in the deep binding pocket of the T monomer and two doxorubicin molecules in a stacked sandwich arrangement in an access pocket at the lateral periplasmic cleft of the L monomer. This access pocket is separated from the deep binding pocket apparent in the T monomer by a switch-loop. The localization and conformational flexibility of this loop seems to be important for large substrates, because a G616N AcrB variant deficient in macrolide transport exhibits an altered conformation within this loop region. Transport seems to be a stepwise process of initial drug uptake in the access pocket of the L monomer and subsequent accommodation of the drug in the deep binding pocket during the L to T transition to the internal deep binding pocket of the T monomer.

Analysis of AcrB and AcrB/DARPin ligand complexes by LILBID MS.

The AcrA/AcrB/TolC complex is responsible for intrinsic multidrug resistance (MDR) in Escherichia coli. Together with the periplasmic adaptor protein AcrA and the outer membrane channel TolC, the inner membrane component AcrB forms an efflux complex that spans both the inner and outer membrane and bridges the periplasm of the Gram-negative cell. Within the entire tripartite complex, homotrimeric AcrB plays a central role in energy transduction and substrate selection. In vitro selected designed ankyrin repeat proteins (DARPin) that specifically bind to the periplasmic domain of AcrB were shown to ameliorate diffraction resolution of AcrB/DARPin protein co-crystals (G. Sennhauser, P. Amstutz, C. Briand, O. Storchenegger, M.G. Grutter, Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors, PLoS Biol 5 (2007) e7). Structural analysis by X-ray crystallography revealed that 2 DARPin molecules were bound to the trimeric AcrB wildtype protein in the crystal, whereas the V612F and G616N AcrB variant crystal structures show 3 DARPin molecules bound to the trimer. These specific stoichiometric differences were analyzed in solution via densitometry after microchannel electrophoresis, analytical ultracentrifugation and via laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS). Using the latter technology, we investigated the gradual disassembly of the AcrB trimer and bound DARPin ligands in dependence on laser intensity in solution. At low laser intensity, the release of the detergent molecule micelle from the AcrB/DARPin complex was observed. By increasing laser intensity, dimeric and monomeric AcrB species with bound DARPin molecules were detected showing the high affinity binding of DARPin to monomeric AcrB species. High laser intensity LILBID MS experiments indicated a spectral shift of the monomeric AcrB peak of 3.1kDa, representing a low molecular weight ligand in all detergent-solubilized AcrB samples and in the AcrB crystal. The identity of this ligand was further investigated using phospholipid analysis of purified AcrB and AcrB variant samples, and indicated the presence of phosphatidylethanolamine and possibly cardiolipin, both constituents of the Escherichia coli membrane.

Structural and functional aspects of the multidrug efflux pump AcrB.

The tripartite efflux system AcrA/AcrB/TolC is the main pump in Escherichia coli for the efflux of multiple antibiotics, dyes, bile salts and detergents. The inner membrane component AcrB is central to substrate recognition and energy transduction and acts as a proton/drug antiporter. Recent structural studies show that homotrimeric AcrB can adopt different monomer conformations representing consecutive states in an allosteric functional rotation transport cycle. The conformational changes create an alternate access drug transport tunnel including a hydrophobic substrate binding pocket in one of the cycle intermediates.

The AcrB efflux pump: conformational cycling and peristalsis lead to multidrug resistance.

Antimicrobial resistance of human pathogenic bacteria is an emerging problem for global public health. This resistance is often associated with the overproduction of membrane transport proteins that are capable to pump chemotherapeutics, antibiotics, detergents, dyes and organic solvents out of the cell. In Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, tripartite multidrug efflux systems extrude a large variety of cytotoxic substances from the cell membrane directly into the medium bypassing the periplasm and the outer membrane. In E. coli, the tripartite efflux system AcrA/AcrB/TolC is the pump in charge of the efflux of multiple antibiotics, dyes, bile salts and detergents. The trimeric outer membrane factor (OMF) TolC forms a beta-barrel pore in the outer membrane and exhibits a long periplasmic alpha-helical conduit. The periplasmic membrane fusion protein (MFP) AcrA serves as a linker between TolC and the trimeric resistance nodulation cell division (RND) pump AcrB, located in the inner membrane acting as a proton/drug antiporter. The newly elucidated asymmetric structure of trimeric AcrB reveals three different monomer conformations representing consecutive states in a transport cycle. The monomers show tunnels with occlusions at different sites leading from the lateral side through the periplasmic porter (pore) domains towards the funnel of the trimer and TolC. The structural changes create a hydrophobic pocket in one monomer, which is not present in the other two monomers. Minocyclin and doxorubicin, both AcrB substrates, specifically bind to this pocket substantiating its role as drug binding pocket. The energy transduction from the proton motive force into drug efflux includes proton binding in (and release from) the transmembrane part. The conformational changes observed within a triad of essential, titratable residues (Asp407/Asp408/Lys940) residing in the hydrophobic transmembrane domain appear to be transduced by transmembrane helix 8 and associated with the conformational changes seen in the periplasmic domain. From the asymmetric structure a possible peristaltic pump transport mechanism based on a functional rotation of the AcrB trimer has been postulated. The novel transport model merges Jardetzky's alternate access pump mechanism with the rotating site catalysis of F(1)F(0) ATPase and suggests a working hypothesis for the transport mechanism of RND transporters in general.

Engineered disulfide bonds support the functional rotation mechanism of multidrug efflux pump AcrB.

The AcrA-AcrB-TolC complex is the major multidrug efflux pump in Escherichia coli. The asymmetric structure of the trimeric inner-membrane component AcrB implies functional rotation of the monomers and a peristaltic mode of drug efflux. This mechanism suggests the occurrence of conformational changes in the periplasmic pore domain through the movements of subdomains during cycling of the monomers through the different states loose (L), tight (T) and open (O). We introduced cysteines at the interfaces of potentially moving subdomains, leading to disulfide bond formation as quantified by alkylation of free cysteines and MALDI-TOF analysis. Inhibition of pump function as a result of cross-linking caused increased susceptibility to noxious compounds and reduction of N-phenylnaphthylamine efflux. Regain of function for impaired mutants was obtained upon exposure to the reducing agent DTT. The results support the presence of the asymmetric AcrB trimer in E. coli membranes and the functional rotation mechanism.

Structural asymmetry of AcrB trimer suggests a peristaltic pump mechanism.

The AcrA/AcrB/TolC complex spans the inner and outer membranes of Escherichia coli and serves as its major drug-resistance pump. Driven by the proton motive force, it mediates the efflux of bile salts, detergents, organic solvents, and many structurally unrelated antibiotics. Here, we report a crystallographic structure of trimeric AcrB determined at 2.9 and 3.0 angstrom resolution in space groups that allow asymmetry of the monomers. This structure reveals three different monomer conformations representing consecutive states in a transport cycle. The structural data imply an alternating access mechanism and a novel peristaltic mode of drug transport by this type of transporter.