Thermodynamics and Unbinding Kinetics of A22 at Multiple Actin-Binding Sites Revealed by Enhanced Sampling Simulations

Abstract The cytoskeletal protein plays a major role in various cellular processes. Understanding the interactions of small molecules with cytoskeletal protein therefore might help in the development of therapeutics. Employing combined molecular docking, all-atom molecular dynamics (MD), and enhanced sampling technique, we studied bacterial inhibitor A22 and actin protein interaction. Five probable A22 binding sites (S1–S5) were observed in actin and unbiased MD simulations of 100 ns to 1000 ns showed structural stability at these sites. Interaction analyses showed A22 to form transient interactions except at sites S1, S3, and S5 where long-lived interactions were present. Enhanced sampling simulations quantitatively estimated ligand dissociation free energy (ΔGb) ∼ −1.3 ± 0.6 kcal/mol to −4.9 ± 1.5 kcal/mol for sites S1–S4 with residence times ∼μs to ms. For site S5, we observed the highest binding affinity with dissociation free energy (ΔGb) ∼ −8.8 ± 3.8 kcal/mol with residence time ∼sec. Analyses of the dissociation trajectories predict multiple dissociation pathways for A22 and found key gatekeeper residues at specific-sites facilitating ligand dissociation. Comparison of A22 with other known inhibitors suggests that A22 binds actin with relatively lower affinity, however, exhibits site-specific unbinding kinetics. Thus, our study provides a detailed mechanistic overview of actin + A22 interaction, its various binding modes, binding affinities, and unbinding kinetics. It also shows the importance and usage of metadynamics and its variants in exploring rare events like ligand–protein interaction. Deeper insights gained from this study expands our understanding of cytoskeletal ligand dynamics. These knowledges will be paramount in designing drug targeting cytoskeletal protein actin.