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Influence of naturally-occurring 5'-pyrophosphate-linked substituents on the binding of adenylic inhibitors to ribonuclease a: an X-ray crystallographic study.

ABSTRACT: Ribonuclease A is the archetype of a functionally diverse superfamily of vertebrate-specific ribonucleases. Inhibitors of its action have potential use in the elucidation of the in vivo roles of these enzymes and in the treatment of pathologies associated therewith. Derivatives of adenosine 5'-pyrophosphate are the most potent nucleotide-based inhibitors known. Here, we use X-ray crystallography to visualize the binding of four naturally-occurring derivatives that contain 5'-pyrophosphate-linked extensions. 5'-ATP binds with the adenine occupying the B(2) subsite in the manner of an RNA substrate but with the gamma-phosphate at the P(1) subsite. Diadenosine triphosphate (Ap(3)A) binds with the adenine in syn conformation, the beta-phosphate as the principal P(1) subsite ligand and without order beyond the gamma-phosphate. NADPH and NADP(+) bind with the adenine stacked against an alternative rotamer of His119, the 2'-phosphate at the P(1) subsite, and without order beyond the 5'-alpha-phosphate. We also present the structure of the complex formed with pyrophosphate ion. The structural data enable existing kinetic data on the binding of these compounds to a variety of ribonucleases to be rationalized and suggest that as the complexity of the 5'-linked extension increases, the need to avoid unfavorable contacts places limitations on the number of possible binding modes.

SUBMITTER: Holloway DE

PROVIDER: S-EPMC2816359 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Influence of naturally-occurring 5'-pyrophosphate-linked substituents on the binding of adenylic inhibitors to ribonuclease a: an X-ray crystallographic study.

Holloway Daniel E DE Chavali Gayatri B GB Leonidas Demetres D DD Baker Matthew D MD Acharya K Ravi KR

Biopolymers 20091201 12

Ribonuclease A is the archetype of a functionally diverse superfamily of vertebrate-specific ribonucleases. Inhibitors of its action have potential use in the elucidation of the in vivo roles of these enzymes and in the treatment of pathologies associated therewith. Derivatives of adenosine 5'-pyrophosphate are the most potent nucleotide-based inhibitors known. Here, we use X-ray crystallography to visualize the binding of four naturally-occurring derivatives that contain 5'-pyrophosphate-linked ...[more]

PMID: 19191310

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Project description: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 B(2) and the placement of the 5'-beta-phosphate of the adenylate (instead of the alpha-phosphate) at subsite P(1) 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'-beta-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 P(1) and B(2). 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 (epsilon(p)) 2-20, in good agreement with the experimental value (-3.6 kcal/mol); the agreement becomes exact with epsilon(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 Lys(7) and Lys(66) of the more remote sites P(2) and P(0), and His(119) of site P(1).

Dataset Information

Influence of naturally-occurring 5'-pyrophosphate-linked substituents on the binding of adenylic inhibitors to ribonuclease a: an X-ray crystallographic study.

Publications

Influence of naturally-occurring 5'-pyrophosphate-linked substituents on the binding of adenylic inhibitors to ribonuclease a: an X-ray crystallographic study.

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