Thermodynamics and unbinding kinetics of A22 at multiple actin binding sites revealed by enhanced sampling simulations

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 infrequent well-tempered metadynamics (iWT-MetaD) 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 - 500 ns showed structural stability at these sites. Interaction analyses showed A22 to mostly forms transient interactions except at sites S4 and S5 where long-lives interactions were present. Enhanced sampling simulations quantitatively estimated ligand dissociation free energy ~ - 2 kcal/mol to -3.5 kcal/mol for sites S1-S4 with residence times ~ microseconds to milliseconds. For site S5, we observed the highest binding affinity (~ - 6.05 kcal/mol) and longest residence time ~ 0.75 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 suggest that A22 binds actin with relatively lower affinity, however, exhibits site-specific unbinding kinetics. Thus, our study provides a detailed mechanistic overview of A22-actin interaction, its various binding modes, and unbinding kinetics. It also shows the importance and usage of infrequent metadynamics 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.