Molecular Dynamics Simulations of the Bacterial Outer Membrane Channels TolC and OprM & dxTuber, a Biomolecular Cavity Detection Tool based on Protein and Solvent Dynamics

Abstract

The multidrug resistance of bacteria is a serious phenomenon in current medical treatment. Beginning with the introduction of antibiotics more and more bacterial strains achieved resistance against these chemical compounds and over the years a competition between antibiotic drug discovery and bacterial drug resistance arose. The well studied Gram-negative bacteria <em>Escherichia coli</em> and <em>Pseudomonas aeruginosa </em>serve in this work as a model organisms for bacterial resistance against antibiotics. Both bacteria evolved multidrug resistant strains through several strategies, including the expelling of harming compounds through efflux systems. The over expression of these efflux systems in the bacterial membranes are responsible for resistance against many antibiotic compounds. <br /> The AcrA/B-TolC efflux system induces resistance of <em>E.coli</em> against a broad range of antibiotics. Ranging from the inner membrane towards the outer membrane, the efflux system spans the entire periplasmic space. The system consists of the inner membrane transporter AcrB, the membrane fusion protein AcrA and the outer membrane channel TolC. TolC itself cooperates with several inner membrane transporters and facilitates the export of harming compounds across the outer membrane. Due to this versatility TolC could become a target of drug treatment. A disabled or blocked TolC could prevent drug extrusion via systems that use TolC as an exit gate. At the time of writing the gating functionality of TolC is not known in detail. To gain insights into TolC functionality two series of unbiased molecular dynamics (MD) simulations were performed. Whereas the first series was carried out in absence of AcrB the second one was executed in presence of the AcrB docking domain (AcrB-DD). <br /> For the first series unbiased MD simulations between 150-300 ns in a Palmitoyloleoylphosphatidylethanolamine (POPE) / NaCl / water environment were calculated. In most of these simulations TolC opens and closes freely on extracellular side hinting at the absence of a gating functionality on this side. On periplasmic side a double aspartate ring restricts substrate passage in all simulations and grasping-like motions were noticed for the tip loops of helix 7 & 8. A consecutive binding of two sodium ions inside the lower periplasmic part of TolC occured in one simulation, which induced a stabilized closed state on periplasmic side. <br /> TolC remained closed on periplasmic side unless all ions were removed from the simulation box indicating a sodium dependent lock on this side. For the second series of MD simulations we added the AcrB-DD to the previously described system setup based on orientations of a previously published data driven modeled structure. Four unbiased 150 ns MD simulations were calculated and in one of these simulations the docking domain spontaneously docks onto TolC. The latter simulation was extended to a simulation time of 1.05 μs resulting in a tighter binding between AcrB and TolC with regards to the modeled structure. A preferred open conformation on extracellular hints analogue to TolC only simulations at the absence of a lock on extracellular side. On the AcrB-facing side TolC's tip loops located at helix 7 & 8 opened up and were stabilized by the AcrB docking domain. However, the double aspartate ring remained closed until the end of the simulation, meaning that either the simulation time is too short to observe an opening of TolC or that another part of the AcrA/B-TolC efflux system is missing to open TolC. <br /> In <em>Pseudomonas aeruginosa</em> OprM had been identified as a TolC homologue protein. OprM is part of the multidrug efflux system MexA/B-OprM and acts as an exit duct for several inner membrane transporters. Also for OprM the gating mechanisms are not known in detail at time of writing. To explore OprM's gating mechanisms it has been simulated in a POPE / NaCl / water environment. During all five 200 ns long MD simulations OprM opens and closes freely on extracellular side suggesting also for OprM the absence of a gating mechanism on extracellular side. The tip loops of helix 7 & 8 on periplasmic side open up in a way comparable to TolC simulations and in contrast to TolC no closing motions were noticed for these helices for OprM. In OprM a single aspartate ring limits substrate passage on the inner membrane facing side of OprM. In contrast to TolC simulations a slight opening of this aspartate ring was measured in all five simulations. The absence of heightened sodium densities near the periplasmic entrance regions could mean that either longer simulation time is needed to observe a sodium induced closure of OprM or that the periplasmic access is regulated only by the aspartate ring. Despite the absence of heightened sodium densities in the aspartate ring region, clear peaks of high sodium densities identified sodium pockets between the equatorial region and the aspartate ring region formed by Asp171 and Asp230. <br /> Voids inside of proteins can indicate substrate binding sites, ion pockets, pathways through channel proteins, their open and closed states and active sites. Over the years numerous cavity detection tools have been introduced to identify and highlight these voids. All available cavity detection tools were based on static structures and present cavities for single protein conformations only.<br /> With dxTuber we developed and introduced a novel cavity detection tool based on an ensemble of protein conformations. It uses averaged protein and solvent density maps, which are derived from MD trajectories, as input. With this technique protein dynamics are taken into account and cavities are detected through the separation of protein external solvent from protein internal solvent. Protein internal solvent can be grouped into cavities and stored in the commonly used PDB file format. Individual cavities can be separated via the atom name field of the PDB file format. dxTuber itself can calculate cavity volume and the cross-sectional area of a single cavity along a principle axis. For convenience a graphical user interface (GUI) and a command line interface (CLI) of dxTuber are released under the GPL v2.