By Wenning Wang
Department of Chemistry, Fudan University

AcrB is the inner membrane transporter of the tripartite multidrug efflux pump AcrAB-TolC in Gram-negative bacteria, which poses a major obstacle to the treatment of bacterial infections. X-ray structures have identified two types of substrate-binding pockets in the porter domains of AcrB trimer: the proximal binding pocket (PBP) and the distal binding pocket (DBP), and suggest a functional rotating mechanism in which each protomer cycles consecutively through three distinct conformational states (access, binding and extrusion). However, the details of substrate binding and translocation between the binding pockets remain elusive. In the first part of this work, we performed atomic simulations to obtain the free energy profile of the translocation of an antibiotic drug doxorubicin (DOX) inside AcrB. Our simulation indicates that DOX binds at the PBP and DBP with comparable affinities in the binding state protomer, and overcomes a 3 kcal/mol energy barrier to transit between them. Obvious conformational changes including closing of the PC1/PC2 cleft and shrinking of the DBP were observed upon DOX binding in the PBP, resulting in an intermediate state between the access and binding states. Taken together, the simulation results reveal a detailed stepwise substrate binding and translocation process in the framework of functional rotating mechanism. We next combined targeted MD, Steered MD simulations and MM-GBSA method to compare the binding and extrusion of DOX and inhibitor molecule ABI in AcrB. Targeted MD simulations show that DOX dissociates from the DBP earlier than ABI, and this is mainly due to the binding of ABI in the hydrophobic trap of the DBP. Consistently, MM-GBSA calculations gave higher binding free energy of ABI than that of DOX in the DBP of the binding monomer, while the binding free energies of the two molecules in the extrusion monomer are nearly equivalent. The PMF profiles of DOX/ABI extrusion from AcrB based on steered MD simulations demonstrate that the extrusion of ABI has to overcome higher energy barrier due to the more evident conformational changes at the exit gate as ABI was exported. Therefore, the inhibitory mechanism of ABI can be ascribed to two aspects: the stronger binding affinity in the DBP and the higher energy barrier required to pass through the exit gate.