Computational Studies of the Escherichia coli Multidrug Efflux Pump AcrAB-TolC

Abstract

When it was known that special substances were able to kill microbes, it was observed that some organisms were unaffected by normally lethal doses. During the last decades, the excessive usage of antibiotics has led to special bacterial strains, resistant against a wide range of antimicrobial drugs. Because of the increasing ineffectiveness of existing pharmaceutical therapies, a detailed insight into the underlying multidrug resistance mechanisms is paramount in order to develop novel strategies to treat resulting infectious diseases.<br /> Key factors for antibiotic resistance in Gram-negative bacteria are efflux pumps of the resistance nodulation division (RND) family, which transports drugs out of the cell before they are able to affect their target. One of the best studied RND efflux systems is the tripartite AcrAB-TolC multidrug efflux pump of Escherichia coli, which was found to be responsible for the tolerance against almost all classes of antibacterial compounds due to an interplay of its components: the inner-membrane transporter AcrB, the periplasmic adaptor protein AcrA, and the outer-membrane channel TolC.<br /> This thesis is meant to gain insights into the conformational and functional properties of AcrAB-TolC in complex via Molecular Dynamics (MD) simulations of the best available structure: a docking model based on biochemical cross-linking data. In this model, the inner-membrane transporter and the outer-membrane channel are forming a continuous tunnel through the entire periplasmic space, by which transported substrates are expelled to the cell exterior. The used MD model considers the protein complex in two parallel glycerophospholipid bilayer models, representing the inner and outer membrane of Escherichia coli. Both a protein model of that size as well as its simulation embedded in two model membranes, renders this thesis as pioneering work in the field of molecular modeling. Thus, problems occur with regard to the established system setup and simulation protocols.<br /> The current state of membrane protein simulations is reviewed with special focus on existing methodologies for the embedding of protein structures into model membranes. Concerning the stability of the complex model, critical issues for the simulation of a protein/twomembrane model, such as problems resulting from the two compartments or the model microenvironment, are discussed. In order to address these issues, two software applications were developed to improve and automate the additional steps of membrane protein embedding in the system setup protocol. To verify the conformation of AcrA in the docked model, the structure of the periplasmic adaptor protein has been completed on its amino-terminus and associated to a membrane via a generated model of the Gram-negative lipoprotein anchor for MD simulations. While the overall behavior of rigid domains and flexible inter-domain regions of AcrA is in line with existing studies and computational data of the adaptor protein, the AcrA conformation drifts apart from the initial configuration of the docked conformer and adopts a structure more similar to the experimentally determined X-ray structure. This result suggests that the conformer in the docked model is in a non-equilibrium state, which renders the validity of the complex model questionable. Looking at AcrA in a fully-protonated state revealed a larger distance between the membrane surface and the structured part of the protein, which might represent a conformation relevant for the assembly of the complex.