Estimating the correct binding modes of ligands in protein–ligand complexes is crucial not only in the drug discovery process but also for elucidating potential toxicity mechanisms. In the current paper, we propose a computational modeling workflow using the combination of docking, classical molecular dynamics (cMD), accelerated molecular dynamics (aMD) and free-energy perturbation (FEP+ protocol) for identification of possible ligand binding modes. It was applied for investigation of selected perfluorocarboxyl acids (PFCAs) in the PPARγ nuclear receptor. Although both regular and induced fit docking failed to reproduce the experimentally determined binding mode of the ligands when docked into a non-native X-ray structure, cMD and aMD simulations successfully identified the most probable binding conformations. Moreover, multiple binding modes were identified for all of these compounds and the shorter-chain PFCAs continuously moved between a few energetically favorable binding conformations. On the basis of MD predictions of binding conformations, we applied the default and also redesigned FEP+ sampling protocols, which accurately reproduced experimental differences in the binding energies. Thus, the preliminary MD simulations can also provide helpful information about correct setup of the FEP+ calculations. These results show that the PFCA binding modes were accurately predicted and that the FEP+ protocol can be used to estimate free energies of binding of flexible ligands that are not typical druglike compounds. Our in silico workflow revealed the specific ligand–residue interactions within the ligand binding domain and the main characteristics of the PFCAs, and it was concluded that these compounds are week PPARγ partial agonists. This work also suggests a common pipeline for identification of ligand binding modes, ligand–protein dynamics description, and relative free-energy calculations.

The N501Y and K417N mutations in spike protein of SARS-CoV-2 and their combination arise questions but the data about their mechanism of action at molecular level is limited. Here, we present Free energy perturbation (FEP) calculations for the interactions of the spike S1 receptor binding domain (RBD) with both the ACE2 receptor and an antibody derived from COVID-19 patients. Our results shown that the S1 RBD-ACE2 interactions were significantly increased whereas those with the STE90-C11 antibody dramatically decreased; about over 100 times. The K417N mutation had much more pronounced effect and in a combination with N501Y fully abolished the antibody effect. This may explain the observed in UK and South Africa more spread of the virus but also raise an important question about the possible human immune response and the success of already available vaccines.

In recent years, the nuclear receptors (NR) dynamics have been studied extensively by various approaches. However, the transition path of helix 12 (H12) to an agonist or an antagonist conformation and the exchange pathway between these states is not clear yet. A number of accelerated molecular dynamics (aMD) runs were performed on both an ERα monomer and a homodimer with a total length of 2.2 μs. We have been able to sample reasonably well the H12 conformational landscape to reproduce precisely both the agonist and the antagonist conformations, starting from an unfolded position, and to describe the transition path between them, even in the presence of an agonist ligand. These conformations were the most prevalent, suggesting that the extended H12 state is not likely to exist and that the natural ERα H12 position might exist in both the agonist and antagonist states. Remarkably, the H12 transition occurs and is regulated only in a dimer form and the proper agonist or antagonist H12 conformation can be achieved solely in one of the dimer subunits. These results clearly demonstrate that clusters of the two well-known H12 states exist by themselves in the protein free energy landscape, i.e. they are not constituted directly by the ligands, and dimerization favors the switch between them. Conversely, in a monomer, no transitions have been observed. Thus, the dimer formation helps the constitution of populations of discrete H12 conformational states and reshapes the conformational landscape. Further analyses have shown that these observations can be explained by specific interface and long range protein–protein interactions, resulting in conformational fluctuations in helices 5 and 11. Based on these results, a new ERα activation/deactivation mechanism and a sequence of binding events during receptor activity modulation have been suggested according to which ligands control the H12 conformation via alterations of the inter-dimer interactions. These findings agree with the HDX and fluorescence experiments and provide an explanation on a structural basis of these data, demonstrating that the dynamics of H12 are not altered greatly upon ligand binding and large fluctuations at the end of H11 are present.