Substrate Specificity Checkpoints of the Multidrug Efflux Pump MexF from <i>Pseudomonas aeruginosa</i>

Multidrug efflux pumps of the resistance-nodulation-division (RND) superfamily are major contributors to antibiotic resistance in <i>Pseudomonas aeruginosa</i>. Among these, the MexEF-OprN system, when overproduced in clinical isolates, confers resistance to fluoroquinolones, trimethoprim, and chloramphenicol. The inner-membrane RND transporter MexF in this complex exhibits a relatively narrow substrate specificity and the molecular mechanisms underlying this specificity are still unclear. Here, we employed a combination of experimental and computational approaches to dissect the role of a major putative recognition/binding site, the Access pocket, in the substrate specificity of MexF. Mutations at four selected positions D132, P136, G626, and S729 altered resistance profiles and substrate specificity in a residue- and substrate-specific manner. Notably, substitutions at P136 enhanced efflux of most tested antibiotics, among which are 21 fluoroquinolones with different structures. Substitutions in S729, on the other hand, either enhanced or severely impaired MexF activity depending on the substitution. Antibiotic substrates were found to compete with a fluorescent probe for MexF efflux revealing overlapping binding determinants and shared translocation paths within the transporter. Ensemble docking and contact frequency analyses further demonstrated that mutations reshaped ligand binding preferences within the periplasmic cleft, modulating the probability of transition to the Deep pocket and subsequent extrusion. Our results demonstrate that MexF is optimized to trimethoprim-like compounds and single substitutions in key residues can dramatically change the substrate spectrum of this pump. These findings underline the importance of not only static binding contacts between substrates and a polyspecific transporter such as MexF but also spatial occupancy and pathway integrity in determining drug efflux efficiency.