# Substrate Specificity Checkpoints of the Multidrug Efflux Pump MexF from Pseudomonas aeruginosa

## TL;DR

This study investigates how the MexF efflux pump in Pseudomonas aeruginosa selectively expels antibiotics, revealing how specific mutations alter its drug resistance profile.

## Contribution

The study identifies key residues in MexF that modulate substrate specificity and efflux efficiency through experimental and computational methods.

## Key findings

- Mutations at P136 increased efflux of multiple fluoroquinolones and other antibiotics.
- Substitutions at S729 had variable effects, either enhancing or impairing MexF activity.
- Antibiotics and a fluorescent probe compete for binding, indicating shared translocation pathways in MexF.

## Abstract

Multidrug efflux
pumps of the resistance-nodulation-division (RND)
superfamily are major contributors to antibiotic resistance in Pseudomonas aeruginosa. 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.