Site-Directed Disulfide Cross-Linking Shows that Cleft Flexibility in the Periplasmic Domain Is Needed for the Multidrug Efflux Pump AcrB of Escherichia coli

Abstract

Escherichia coli AcrB is a multidrug efflux transporter that recognizes multiple toxic chemicals having diverse structures. Recent crystallographic studies of the asymmetric trimer of AcrB suggest that each protomer in the trimeric assembly goes through a cycle of conformational changes during drug export. However, biochemical evidence for these conformational changes has not been provided previously. In this study, we took advantage of the observation that the external large cleft in the periplasmic domain of AcrB appears to become closed in the crystal structure of one of the three protomers, and we carried out in vivo cross-linking between cysteine residues introduced by site-directed mutagenesis on both sides of the cleft, as well as at the interface between the periplasmic domains of the AcrB trimer. Double-cysteine mutants with mutations in the cleft or the interface were inactive. The possibility that this was due to the formation of disulfide bonds was suggested by the restoration of transport activity of the cleft mutants in a dsbA strain, which had diminished activity to form disulfide bonds in the periplasm. Furthermore, rapidly reacting, sulfhydryl-specific chemical cross-linkers, methanethiosulfonates, inactivated the AcrB transporter with double-cysteine residues in the cleft expressed in dsbA cells, and this inactivation could be observed within a few seconds after the addition of a cross-linker in real time by increased ethidium influx into the cells. These observations indicate that conformational changes, including the closure of the external cleft in the periplasmic domain, are required for drug transport by AcrB.It is now well known that multidrug efflux transporters cause problems in cancer chemotherapy, as well as in antibiotic treatment of bacterial infections. These transporters recognize many structurally dissimilar toxic compounds and actively extrude them from the cell. The Escherichia coli AcrB transporter (14, 15), which belongs to the resistance-nodulationdivision family (32), is responsible for most of the intrinsic drug resistance of this organism (20,21,28) and is also perhaps the best-studied bacterial multidrug pump. It is a homotrimer in which each subunit contains 12 transmembrane (TM) helices and two large periplasmic domains, one between TM helix 1 (TM1) and TM2 and one between TM7 and TM8. The trimeric AcrB molecule in turn occurs as a multiprotein complex (30,31,36) together with the outer membrane channel protein TolC (8, 12) and the periplasmic linker protein AcrA (14). The top of the periplasmic domain of AcrB is thought to interact with the internal end of TolC, and this structure allows direct export of drugs to the external medium (20).AcrB utilizes the proton motive force as energy for its transport function (14,34,35). Charged residues within the TM helices of this transporter, including Asp407, Asp408, and Lys940, have been identified as amino acid residues essential for activity (19), presumably functioning as components of the proton relay system. We previously...

Multidrug binding properties of the AcrB efflux pump characterized by molecular dynamics simulations

“…On the basis of these findings, a functional rotation hypothesis was suggested, in which each protomer assumes successively one of the aforementioned conformations (9)(10)(11). This idea has been supported by several biochemical studies (12)(13)(14), as well as by molecular simulation (15,16). It thus seems that this distal binding pocket (Fig.…”

Mechanism of recognition of compounds of diverse structures by the multidrug efflux pump AcrB ofEscherichia coli

“…(i) Unlike the previously reported sites that were wide open, this site is surrounded on most sides, except an opening into the tunnel (Fig. 2C). (ii) Only one protomer among the three, the Binding protomer, binds the drug molecule in this pocket, and this led to the proposal of the functionally rotating mechanism of transport (21-23), which was confirmed by biochemical studies (24)(25)(26). (iii) The open pocket of this binding site collapses in the Extrusion protomer, in which the path to the central funnel opens up.…”

“…The potential competitor was added, and 5 min later fluorescein maleimide was added to 20 or 40 μM, and the labeling was terminated after 30 min at room temperature by the addition of DTT to 10 mM. Cells were centrifuged, washed twice with 10 mM Hepes-KOH, broken by sonication in the presence of Complete, EDTA-free Protease Inhibitor Mixture (Roche), and the linked AcrB trimers with the C-terminal His-tag were isolated with Co 2+ -TALON resin essentially as described (24). The protein was analyzed by SDS/PAGE using 7.5% acrylamide after the removal of imidazole by gel filtration with Biogel P-6DG.…”

Kinetic behavior of the major multidrug efflux pump AcrB of Escherichia coli

“…This content was determined by Western blot analysis (35), by using a standard curve constructed by using known amounts of purified AcrB protein.…”

“…Linear gradient plates containing nitrocefin in the bottom layer were made with LB agar in square Petri dishes, and bacterial suspensions diluted to an OD660 of 0.1 were used as inoculum, as described earlier (35). Plates were observed after overnight incubation at 37°C.…”