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

Abstract

Multidrug resistance in Gram-negative bacteria, to which multidrug efflux pumps such as the AcrB transporter makes a major contribution, is becoming a major public health problem. Unfortunately only a few compounds have been cocrystallized with AcrB, and thus computational approaches are essential in elucidating the interaction between diverse ligands and the pump protein. We used molecular dynamics simulation to examine the binding of nine substrates, two inhibitors, and two nonsubstrates to the distal binding pocket of AcrB, identified earlier by X-ray crystallography. This approach gave us more realistic views of the binding than the previously used docking approach, as the explicit water molecules contributed to the process and the flexible binding site was often seen to undergo large structural changes. We analyzed the interaction in detail in terms of the binding energy, hydrophobic surface-matching, and the residues involved in the process. We found that all substrates tested bound to the pocket, whereas the binding to this site was not preferred for the nonsubstrates. Interestingly, both inhibitors [Phe-Arg-β-naphthylamide and 1-(1-naphtylmethyl)-piperazine] tended to move out of the pocket at least partially, getting into contact with a glycine-rich loop that separates the distal pocket from the more proximal region of the protein and is thought to control the access of substrates to the distal pocket.bacterial drug efflux pump | drug-binding pocket | efflux inhibitors

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

“…Side chains of other most relevant residues are shown with thinner sticks, colored by residue type and red in wild type and G288D, respectively. The orientation of CIP is derived from the MD simulations (27), and that of DOXO is based on the experimental X-ray structure as from PDB ID code 4DX7 (22). Lines denote values, for strains carrying the G288D mutation, which are significantly different to the strain carrying the wild-type AcrB.…”

“…Because this site has been shown to be extremely adaptable to many substrates (27), it cannot be ruled out that despite the large effect due to G288D substitution the pocket would still be able to accommodate most compounds but with altered affinities. In agreement with such an interpretation we observed that the G288D substitution caused an increase in intracellular accumulation of doxorubicin and minocycline, which have been reported to bind to the phenylalanine cluster in the distal binding pocket (2), and concomitant increased susceptibility.…”

“…Full investigation of the interaction between drugs and the mutated protein clearly merits a systematic, stand-alone full MD simulation (e.g., ref. 27) and/or a crystallographic solution (e.g., ref. 30).…”

“…Subsequently, MD simulations in explicit solvent were carried out to identify major differences in structure and dynamics of the distal binding pocket. A tested, reduced model of the S. Typhimurium AcrB containing only the periplasmic domains was used as in previous computational studies on E. coli AcrB (27)(28)(29).…”

“…Consistent with MIC and drug accumulation data below, and based on the assumption that the binding of tetra-ring structures follows that in the cocrystal structures, the G288D substitution would result in pronounced steric clashes with both doxorubicin and minocycline, due also to displacement of amino acid F178 discussed above. Although an experimental structure of the ciprofloxacin-AcrB complex is lacking, a recent computational study (27) suggested that it binds to the bottom of the distal pocket, in a different position than doxorubicin (Fig. 3).…”