Conformational Transitions and Proton Conduction in the Multidrug Efflux Pump AcrB

Abstract

The increasing problem of multi-drug resistance (MDR) in cancer therapy or bacterial infections is to a large degree caused by multi-drug efflux pumps in pathogenic cells. In Escherichia coli, a major resistance mechanism against antibiotics is based on a tripartite multi-drug export complex comprising the inner membrane translocase AcrB, the membrane-fusion protein AcrA and the outer-membrane channel TolC. AcrB functions as the engine of this complex, using proton motive force to expel a wide variety of unrelated toxic compounds such as antibiotics, disinfectants or detergents. The molecular mechanisms of how proton conduction through AcrB is coupled to drug expulsion are not fully understood yet. Here we report a combination of normal mode analysis (NMA) and molecular dynamics (MD) simulation to investigate conformational transitions occurring in the AcrB reaction cycle and to identify residues crucial for proton conduction. In the crystallographic structure of AcrB each monomer is trapped in a different conformation, representing consecutive states in the transport mechanism. Applying the elastic network NMA variant of minimum action pathway (Kim et al. 2002), we computed transitions between these states. The resulting c-alpha trajectories were then converted back to all atom in an approach of steered energy minimization. We also performed multi-copy MD simulations of AcrB embedded in a phospholipid/water environment using the GroMACS simulation package. Mapping the proton conduction pathway was done on the basis of protein-internal water dynamics and monitoring their frequency of forming hydrogen bonds to adjacent residues.