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

Abstract

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.

Conserved small protein associates with the multidrug efflux pump AcrB and differentially affects antibiotic resistance

“…Recent studies on the crystal structure of substrate bound AcrB (28)(29)(30) and biochemical mapping of the substrate path (31,32) have shown that there are multiple drug-binding sites in the large periplasmic domain of AcrB. We propose that AcrZ binding to AcrB could trigger conformational changes in the periplasmic domain, thus directly affecting the recognition and capture of certain substrates, such as substrates with lower hydrophobicity (Fig.…”

AcrB drug-binding pocket substitution confers clinically relevant resistance and altered substrate specificity

“…1). This in turn could affect the affinity to various drugs such as doxorubicin and minocycline whose binding positions have been identified by cocrystallization experiments (22) (Fig. 1).…”

“…In the available crystal structures of AcrB in its apo-(PDB ID code 2J8S) (24) and in doxorubicin-(PDB ID code 4DX7) and minocycline-bound forms (PDB ID code 4DX5) (22), G288 is located in the middle of a beta strand, with the neighboring side chains pointing away from the pocket, suggesting that the substituted side chain will protrude into the pocket cavity. Hence, this bulky and charged aspartate residue will have a drastic impact on structure and Experiments were carried out on at least three separate occasions and the mode value is presented.…”

“…Drug binding within AcrB is complex (20,21) and is proposed to include several possible sites before extrusion including a vestibule and proximal and distal binding pocket. It has been suggested that low-molecular-mass compounds could bind directly to the phenylalanine-rich distal binding pocket (5,20,22), bypassing the proximal binding pocket and vestibule, which is (23) implicated in the recognition of larger compounds such as erythromycin (4,5).…”

Anticancer Drugs as Prospective Efflux Pump Inhibitors for Salmonella Typhi Produce Conflicting Results in in Silico and in Vitro Studies

“…5). A number of efflux pump inhibitors phenylalanine-arginine β-naphthylamide (PAβN) [26][27], 1-1-Naphthylmethyl-piperazine (NMP), pyridopyrimidine one and pyrazolopyridines) exerted their inhibitory activity against bacterial efflux pump at concentration range from 70 µg/ml [28] to 100 µg/ml [29], for example, PAβN registered half maximum activity approximately at 70 µg/ml whereas NMP caused EtBr accumulation at 100 µg/ml. An EPI (such as NMP) can also act as antibacterial agent at four-fold (400 µg/ml) concentration to its normal EPI concentration.…”