Recognition of ribonuclease A by 3'-5'-pyrophosphate-linked dinucleotide inhibitors: a molecular dynamics/continuum electrostatics analysis

The proteins of the pancreatic ribonuclease A (RNase A) family catalyze the cleavage of the RNA polymer chain. The development of RNase inhibitors is of significant interest, as some of these compounds may have a therapeutic effect in pathological conditions associated with these proteins. The most potent low molecular weight inhibitor of RNase reported to date is the compound 5′-phospho-2′-deoxyuridine-3-pyrophosphate (P→5)-adenosine-3-phosphate (pdUppA-3′-p). The 3′,5′-pyrophosphate group of this compound increases its affinity and introduces structural features which seem to be unique in pyrophosphate-containing ligands bound to RNase A, such as the adoption of a syn conformation by the adenosine base at RNase subsite B₂ and the placement of the 5′-β-phosphate of the adenylate (instead of the α-phosphate) at subsite P₁ where the phosphodiester bond cleavage occurs. In this work, we study by multi-ns molecular dynamics simulations the structural properties of RNase A complexes with the ligand pdUppA-3′-p and the related weaker inhibitor dUppA, which lacks the 3′ and 5′ terminal phosphate groups of pdUppA-3′-p. The simulations show that the adenylate 5′-β-phosphate binding position and the adenosine syn orientation constitute robust structural features in both complexes, stabilized by persistent interactions with specific active-site residues of subsites P₁ and B₂. The simulation structures are used in conjunction with a continuum-electrostatics (Poisson-Boltzmann) model, to evaluate the relative binding affinity of the two complexes. The computed relative affinity of pdUppA-3′-p varies between −7.9 kcal/mol and −2.8 kcal/mol for a range of protein/ligand dielectric constants (ε_p) 2–20, in good agreement with the experimental value (−3.6 kcal/mol); the agreement becomes exact with ε_p = 8. The success of the continuum-electrostatics model suggests that the differences in affinity of the two ligands originate mainly from electrostatic interactions. A residue decomposition of the electrostatic free energies shows that the terminal phosphate groups of pdUppA-3′-p make increased interactions with residues Lys⁷ and Lys⁶⁶ of the more remote sites P₂ and P₀, and His¹¹⁹ of site P₁.