Computational insights into pH effects on protein conformation, membrane curvature, and water permeation in the E. coli multidrug efflux transporter MdfA

Multidrug resistance (MDR) in bacteria poses a major public health challenge, driven largely by efflux transporters such as the Escherichia coli multidrug transporter MdfA. As a proton-coupled antiporter belonging to the Major Facilitator Superfamily (MFS), MdfA harnesses the transmembrane proton gradient to expel structurally diverse toxic compounds. However, the molecular basis by which environmental pH modulates its conformational adaptability, membrane interactions, and transport efficiency remains poorly understood. In this study, all-atom molecular dynamics simulations were employed to explore the structural and functional response of MdfA within a realistic lipid bilayer under acidic (pH 4.5), near-neutral (pH 6.0), and alkaline (pH 7.5) conditions. Comparative analyses of RMSD, RMSF, radius of gyration, solvent-accessible surface area, dihedral flipping, free energy landscapes, secondary structure evolution, membrane curvature, pore radius, and water permeation collectively revealed a tightly coordinated pH-dependent regulation of transporter behavior. At acidic pH, protonation of acidic residues stabilized an inward-closed, compact conformation characterized by minimal hydration and high membrane curvature, conserving structural integrity but limiting substrate flux. Near-physiological pH 6.0 yielded an optimal balance between rigidity and flexibility, where partial hydration and moderate curvature facilitated dynamic transitions essential for efficient drug efflux. Conversely, at alkaline pH, deprotonation led to helical loosening, increased pore radius, excessive water influx, and structural destabilization, enhancing turnover but reducing stability. These findings delineate a mechanistic continuum linking protonation state to conformational equilibrium, hydration dynamics, and membrane remodeling. By demonstrating that physiological pH optimally tunes MdfA’s structural and energetic landscape for substrate transport, this study provides critical molecular insights into efflux pump adaptability and identifies exploitable pH-dependent vulnerabilities for designing next-generation antibacterial inhibitors targeting multidrug resistance.