Computational studies of AcrB, a Multidrug Efflux Pump

Abstract

AcrA-AcrB-TolC plays a major role in drug resistance in Escherichia coli by extruding bile salts, detergents, organic solvents and many antibiotics that are not structurally related. AcrB, the core of this machinery, has been shown to be functional as an asymmetrical trimer, each protomer of which adopts a conformational state designated as loose (L), tight (T) and open (O) in the cycle of drug transport. Proton translocation has been shown to happen in the transmembrane domain where there are the three titratable amino acids D407, D408 and K940. Mutagenesis studies have shown that these three amino acids serve as the transient station for protons. In the present thesis we used molecular simulations to study the proton translocation across the transmembrane domain. At first, the pKa values of D407, D408 and K940 were predicted using Poisson-Boltzmann calculations and alchemical free energy perturbation simulations. The results show how many protons there are in the transmembrane domain of each state. From the conventional Molecular Dynamics simulations, different water wires were found in the transmembrane domains of the L, T and O state. These water wires revealed the possible path for proton across the transmembrane domains of AcrB. In the analyses of crystal structures, unique structural elements were identified both in the periplasmic and transmembrane domain. By calculating the clashes between protomers in several hypothetical trimers, a scheme of possible state transition pathways is proposed for AcrB working under different drug concentrations. Combining with the former structural studies, computational studies and crystal structure analyses in the current thesis, a global working hypothesis of substrate/proton transport is proposed for AcrB.