Insights into the Inhibitory Mechanism of D13-9001 to the Multidrug Transporter AcrB through Molecular Dynamics Simulations.

The resistance-nodulation-cell division transporter AcrB is responsible for energy transduction and substrate recognition in the tripartite AcrAB-TolC efflux system in Escherichia coli. Despite a broad substrate specificity, only a few compounds have been cocrystallized with AcrB inside the distal binding pocket (DBP), including doxorubicin (DOX) and D13-9001. D13-9001 is a promising efflux pump inhibitor that potentiates the efficacy of a wide variety of antibiotics. To understand its inhibition effect under the framework of functional rotating mechanism, we performed targeted and steered molecular dynamics simulations to compare the binding and extrusion processes of this inhibitor and the substrate DOX in AcrB. The results demonstrate that, with respect to DOX, the interaction of D13-9001 with the hydrophobic trap results in delayed disassociation from the DBP. Notably, the detachment of D13-9001 is tightly correlated with the side-chain reorientation of Phe628 and large-scale displacement of Tyr327. Furthermore, the inhibitor induces much more significant conformational changes at the exit gate than DOX does, thereby causing higher energy cost for extrusion and contributing to the inhibitory effect in addition to the tight binding at DBP.