Exploring the conformation-dependent reactivity and dynamics of a dinucleotide inhibitor of ribonuclease A

The dinucleotide molecule, 5'-phospho-2'-deoxyuridine-3'-pyrophosphate adenosine-3'-phosphate (pdUppA-3'-p) (PUA), is known to act as a highly potent inhibitor of bovine pancreatic ribonuclease A (RNase A) with K_i = 27 nM (D. D. Leonidas et al., Biochemistry, 1999, 38, 10287-10297). In the present work, PUA is investigated to study the effect of different conformations on its residence time and dynamical interactions at the active site of RNase A. Two conformers of PUA, designated as closed and open in terms of their end-to-end distances, have been screened in different environments based on a wide range of global and local reactivity parameters computed using conceptual density functional theory (CDFT). In all the cases studied, the open conformation is found to be more flexible with its reactive sites predominantly localized at the terminal uracil nucleobase. The closed conformer, on the other hand, is associated more with intramolecular hydrogen bonding and van der Waals interactions at both nucleobase termini. When subjected to classical molecular dynamics (MD) simulation, the open conformer is found to dissociate rapidly from the active site of RNase A within 24-300 ns, while the closed conformer remained bound throughout the 5 µs long MD trajectory. The transition from open to closed conformation is found to stabilize the PUA-RNase A complex, lowering its free energy by nearly 25 kcal mol^-1. The crystal structure of the PUA-RNase A complex reports the ligand molecule only in its open conformation at the active site of RNase A (N. Russo and R. Shapiro, J. Biol. Chem., 1999, 274, 14902-14908). The closed conformer has not been detected experimentally as yet. Our results therefore indicate a hereto unexplored role of the closed conformation in the inhibition of RNase A by PUA.