Molecular rationale for the impairment of the MexAB-OprM efflux pump by a single mutation in MexA.

Efflux pumps of the Resistance-Nodulation-cell Division (RND) superfamily contribute to intrinsic and acquired resistance in Gram-negative pathogens by expelling chemically unrelated antibiotics with high efficiency. They are tripartite systems constituted by an inner-membrane-anchored transporter, an outer membrane factor protein, and a membrane fusion protein. Multimerization of the membrane fusion protein is an essential prerequisite for full functionality of these efflux pumps. In this work, we employed complementary computational techniques to investigate the stability of a dimeric unit of MexA (the membrane fusion protein of the MexAB-OprM RND efflux pump of ), and to provide a molecular rationale for the effect of the G72S substitution, which affects MexAB-OprM functionality by impairing the assembly of MexA. Our findings indicate that: i) dimers of this protein are stable in multiple µs-long molecular dynamics simulations; ii) the mutation drastically alters the conformational equilibrium of MexA, favouring a collapsed conformation that is unlikely to form dimers or higher order assemblies. Unveiling the mechanistic aspects underlying large conformational distortions induced by minor sequence changes is informative to efforts at interfering with the activity of this elusive bacterial weapon. In this respect, our work further confirms how molecular simulations can give important contribution and useful insights to characterize the mechanism of highly complex biological systems.