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.
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