Flexibility of Free and AcrB-Bound AcrA in the AcrAB-TolC Multidrug Efflux Pump of Escherichia coli Determined using 3D PMFs

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Key components of the emerging global epidemic of antibiotic resistance are the multidrug efflux pumps of pathogenic, Gram-negative bacteria, which span the periplasmic region between the inner and outer membranes of the cell. These protein complexes are an innate resistance mechanism, removing harmful antibiotics from bacteria. Until 2014 many aspects of the mechanisms for pump activity remained elusive due to the lack of structural data. Since then several increasingly detailed electron microscopy maps of an entire efflux pump complex, AcrAB-TolC, have been resolved, resulting in atomic-level structural models. Using these new models, we performed molecular dynamics simulations to study one of the key components of the protein complex, AcrA, which connects the inner-membrane-bound AcrB to the outer-membrane-bound TolC. We determined the flexibility of free AcrA by calculating a three-dimensional potential of mean force (PMF) focused on three angles that govern AcrA's conformational dynamics. AcrA shows a wide range of accessible orientations, with two main energy basins separated by a low (< 4 kT) barrier, consistent with previously reported equilibrium simulations. The conformation in one of the basins is similar to the AcrB-bound state, suggesting that assembly proceeds by conformational selection. Additional 2D PMFs of AcrA monomers bound to AcrB reveal differences in flexibility at the two distinct binding sites on AcrB, similar to the TriABC efflux pump, which contains a heterodimer membrane fusion protein where the two subunits exhibit unique properties and play different roles in efflux activity (Weeks et al. Mol. Micriobio. 2015). Lastly, equilibrium simulations of wild-type and mutant AcrA monomers elucidate the role of its two terminal domains in pump assembly.