Dynamics of the conformation dependent unbinding of an inhibitor of ribonuclease A

Extensive classical molecular dynamics simulations are reported on the binding/unbinding dynamics of different conformations of the inhibitor, 5 ′ -phospho- 2 ′ -deoxyuridine-3 ′ -pyrophosphate adenosine-3 ′ - phosphate (pdUppA-3 ′ -p or PUA), of the enzyme ribonuclease A (RNase A). The PUA molecule is initially docked at the active site of RNase A in three distinct conformations labeled as open-1 (extracted from PDB id: 1QHC Leonidas et al , Biochemistry 1999, 38, 10287–10297), open-2 , and closed . While the closed system is found to remain bound at the active site till 5 μ s , both the open systems dissociated within 300 n s and went on to explore several surface exposed binding sites transiently. A total of 5 such surface exposed binding sites have been identified from the high dimensional free energy hypersurfaces of the open systems with rate constants of binding and unbinding ranging between 1 0 7 − 1 0 6 s − 1 . Some of these binding sites closely resemble those predicted using deep learning based calculations and corroborate earlier experimental evidence on allosteric inhibition of RNase A by polyphenol based molecules (Ghosh et al , Proteins: Structure, Function, and Bioinformatics 2007, 69, 566–580). The 4 stable states associated with the underlying free energy hypersurface and dynamical correlation of PUA with key amino acid residues reveal new mechanistic insight into the preferential stabilization of the closed conformation at the active site. • MD simulations of the dynamics, free energy and kinetics of binding–unbinding of three distinct conformers of an inhibitor of at multiple sites of RNase A. • Detailed analysis of the preferential stabilization of a closed conformer bound at the active site pocket, not observed in experiments. • Rapid dissociation of open conformations from the active site cavity followed by transient binding at surface sites.