title: Stepwise substrate translocation mechanism revealed by free energy calculations of doxorubicin in the multidrug transporter AcrB.
abstract: AcrB is the inner membrane transporter of the tripartite multidrug efflux pump AcrAB-TolC in E. coli, 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 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.
authors.affiliation: Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Chemistry.
authors.forename: Zhicheng
authors.initials: Z
authors.lastname: Zuo
authors.affiliation: Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Chemistry.
authors.forename: Beibei
authors.initials: B
authors.lastname: Wang
authors.affiliation: Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Chemistry.
authors.forename: Jingwei
authors.initials: J
authors.lastname: Weng
authors.affiliation: Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Chemistry.|Institutes of Biomedical Sciences, Fudan University, Shanghai, P.R. China.
authors.forename: Wenning
authors.initials: W
authors.lastname: Wang
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001665:Binding Sites; D004317:Doxorubicin / Q000494:pharmacology*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D062105:Molecular Docking Simulation; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D000903:Antibiotics, Antineoplastic; D004317:Doxorubicin; D029968:Escherichia coli Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C083496:AcrB protein, E coli