Exploration of Free Energy Surfaces Across a Membrane Channel Using Metadynamics and Umbrella Sampling

Abstract

To reach their site of action, it is essential for antibiotic molecules to cross the bacterial outer membrane. The progress of enhanced sampling techniques in molecular dynamics simulations enables us to understand these translocations at an atomic level. To this end, calculations of free energy surfaces for these permeation processes are of key importance. Herein, we investigate the translocation of a variety of anionic solutes through the outer membrane pore OprO of the Gram-negative bacterium Pseudomonas aeruginosa using the metadynamics and umbrella sampling techniques at the all-atom level. Free energy calculations have been performed employing these two distinct methods in order to illustrate the difference in computed free energies, if any. The investigated solutes range from a single atomic chloride ion over a multiatomic monophosphate ion to a more bulky fosmidomycin antibiotic. The role of complexity of the permeating solutes in estimating accurate free energy profiles is demonstrated by performing extensive convergence analysis. For simple monatomic ions, good agreement between the well-tempered metadynamics and the umbrella sampling approaches is achieved, while for the permeation of the monophosphate ion differences start to appear. In the case of larger molecules such as fosmidomycin it is a tough challenge to achieve converged free energy profiles. This issue is mainly due to neglecting orthogonal degrees of freedom during the free energy calculations. Nevertheless, the freely driven metadynamics approach leads to clearly advantageous results. Additionally, atomistic insights of the translocation mechanisms of all three solutes are discussed.

Fosmidomycin transport through the phosphate‐specific porins OprO and OprP of Pseudomonas aeruginosa

“…This was performed via porin reconstitution into artificial bilayers and electrophysiological studies of anion transport. All experiments performed in presence of fosmidomycin demonstrate a decrease of chloride flux in dependence of fosmidomycin concentration (see Figures 2–4) which suggest, together with the published MD simulations (Golla et al., 2020), that the antibiotic can bind inside and permeate through OprO and OprP in the outer membrane of P. aeruginosa . Fosmidomycin showed already promising results in the treatment of bacterial and plasmodium infections (Lienau et al., 2019; Mine et al., 1980; Quinn et al, 2016).…”

“…The wider constriction of OprO is definitely the reason that fosmidomycin should be able to flip better in order to permeate this pore (Golla et al. 2020). Interestingly, the concentrations for the half saturation of fosmidomycin inhibition of current through OprO, are about three orders of magnitude higher than its MIC, 17.09 μM measured in mice infections of P. aeruginosa (Mine et al., 1980).…”

“…This strengthens the correlation that we quantified between the chosen porins and fosmidomycin. Golla and colleagues have already computed that the antibiotic can go through OprO clarifying entirely its path (Golla et al., 2020). When we compare this with our results of OprP, it is clear that fosmidomycin is able to permeate the OM of P. aeruginosa through OprO and OprP at a high rate.…”

“…We measured the inhibition of chloride transport through OprP and OprO by fosmidomycin in single‐channel and titration experiments following the reconstitutions of the porins in artificial lipid bilayer membranes. Golla and colleagues computed in a recent paper the path of fosmidomycin transport through OprO using molecular dynamics simulations (Golla et al., 2020). Our data are discussed in the light of their results.…”

Natural Polyphenols Inhibit the Dimerization of the SARS-CoV-2 Main Protease: The Case of Fortunellin and Its Structural Analogs

“…The extensive sampling in trajectories of classical MD (60 μs for the monomer and +10 μs for the homodimer) was necessary in order to generate the required large number of conformations for MSM validity [ 25 ]. Due to the large system size, extensive sampling is also important for the enhanced sampling methods to reach convergence for the associated free energy surfaces (FES) of the probed configurational space of 3CL-Pro [ 26 ]. In fact, we observed that the associated FES does not change after 8 μs of sampling at the PTmetaD-WTE level is reached.…”