Drug Uptake Pathways of Multidrug Transporter AcrB Studied by Molecular Simulations and Site-Directed Mutagenesis Experiments

Abstract

Multidrug resistance has been a critical issue in current chemotherapy. In Escherichia coli , a major efflux pump responsible for the multidrug resistance contains a transporter AcrB. Crystallographic studies and mutational assays of AcrB provided much of structural and overall functional insights, which led to the functionally rotating mechanism. However, the drug uptake pathways are somewhat controversial because at least two possible pathways, the vestibule and the cleft paths, were suggested. Here, combining molecular simulations and site-directed mutagenesis experiments, we addressed the uptake mechanism finding that the drug uptake pathways can be significantly different depending on the properties of drugs. First, in the computational free energy analysis of drug movements along AcrB tunnels, we found a ligand-dependent drug uptake mechanism. With the same molecular sizes, drugs that are both strongly hydrophobic and lipophilic were preferentially taken in via the vestibule path, while other drugs favored the cleft path. Second, direct simulations realized totally about 3500 events of drug uptake by AcrB for a broad range of drug property. These simulations confirmed the ligand-dependent drug uptake and further suggested that a smaller drug favors the vestibule path, while a larger one is taken in via the cleft path. Moreover, the direct simulations identified an alternative uptake path which is not visible in the crystal structure. Third, site-directed mutagenesis of AcrB in E. coli verified that mutations of residues located along the newly identified path significantly reduced the efflux efficiency, supporting its relevance in in vivo function.

Evidence of a Substrate-Discriminating Entrance Channel in the Lower Porter Domain of the Multidrug Resistance Efflux Pump AcrB

“…The study proposes two different possible pathways for the translocation of lower-molecular-weight drugs, both using the vestibule entrance channel. I671 is shown as a residue belonging to the front and the back vestibule and I38 as a residue of the back vestibule (21). From the same study, as well as those from other authors, it is supposed that not only the molecular size but also further properties (e.g., lipophilicity and the polar surface area) might be critical for the transport of substrates and the translocation pathways used in RND efflux transporters (16).…”

“…However, it is believed that these substrates pass through the PBP without specific binding to reach their binding site within the DBP (21,22). Several mutations compromising the resistance to drugs of lower molecular weight were described within the DBP (17), and minocycline has been found trapped there in the binding state protomer of cocrystallized AcrB (12).…”

“…Data predominantly from crystallization and computer modeling studies have suggested a vestibule and a cleft entrance. The vestibule channel appears to be opened to the so-called "groove" between TM8 and TM9 and is thought to trap substrates from the outer leaflet of the inner membrane, whereas the cleft pathway is thought to allow the entrance of substrates from the periplasm between subdomains PC1 and PC2 further apart from the upper ending of the TM domain (9,(12)(13)(14)(20)(21)(22). A third option, an entrance on the back side of AcrB open to the central cavity built from the intermolecular space between the protomers is considered to be less likely (13,21).…”

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

“…Thus, our modeling provided a plausible mechanistic explanation for the effect of the mutation G288D. A recent study using MD simulations has highlighted an unexpectedly complex pathway of drug expulsion, with several possible routes of export through AcrB (23). Full investigation of the interaction between drugs and the mutated protein clearly merits a systematic, stand-alone full MD simulation (e.g., ref.…”

Random Mutagenesis of the Multidrug Transporter AcrB from Escherichia coli for Identification of Putative Target Residues of Efflux Pump Inhibitors

“…In addition, recent studies have discovered an access (proximal) pocket separated from the distal binding pocket by a so-called "switch-loop." This is supposed to be another important part of the drug pathway through the AcrB protomer (18,19). The AcrAB-TolC complex is constitutively expressed in E. coli, and overexpression is inducible by drug exposure (20,21), whereas the homologous E. coli transport systems AcrEF and YhiUV have been found to be expressed in vitro in AcrB-deficient E. coli strains only after several selection steps (22,23).…”