Molecular basis for nucleotide-binding specificity: role of the exocyclic amino group 'N2' in recognition by a guanylyl-ribonuclease
| Autor: | William R. Kearney, Kenneth P. Murphy, Travis T. Waldron, Mitchell A. Timmons, S. Ramaswamy, Greta L. Schrift |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
RNase P
Stereochemistry Guanosine Monophosphate Molecular Conformation Guanosine Crystallography X-Ray Substrate Specificity chemistry.chemical_compound Ribonucleases Structural Biology Inosine Monophosphate Nucleotide Ribonuclease Molecular Biology Binding selectivity chemistry.chemical_classification Guanine binding Binding Sites biology Nucleotides Streptomyces aureofaciens Isothermal titration calorimetry Ligand (biochemistry) Isoenzymes Biochemistry chemistry biology.protein Thermodynamics Protein Binding |
| Zdroj: | Journal of molecular biology. 355(1) |
| ISSN: | 0022-2836 |
| Popis: | Proteins interact with nucleotides to perform a multitude of functions within cells. These interactions are highly specific; however, the molecular basis for this specificity is not well understood. To identify factors critical for protein-guanine nucleotide recognition the binding of two closely related ligands, guanosine 3'-monophosphate (3'GMP) and inosine 3'-monophosphate (3'IMP), to Ribonuclease Sa (RNase Sa), a small, guanylyl-endoribonuclease from Streptomyces aureofaciens, was compared using isothermal titration calorimetry, NMR, X-ray crystallography and molecular dynamics simulations. This comparison has allowed for the determination of the contribution of the exocyclic amino group "N2" of the guanine base to nucleotide binding specificity. Calorimetric measurements indicate that RNase Sa has a higher affinity for 3'GMP (K=(1.5+/-0.2)x10(5)) over 3'IMP (K=(3.1+/-0.2)x10(4)) emphasizing the importance of N2 as a key determinant of RNase Sa guanine binding specificity. This result was unexpected as the published structural data for RNase Sa in complex with 3'GMP showed only a potential long-range interaction (3.3A) between N2 and the side-chain of Glu41 of RNase Sa. The observed difference in affinity is largely due to a reduction in the favorable enthalpy change by 10 kJ/mol for 3'IMP binding as compared to 3'GMP that is accompanied by a significant difference in the heat capacity changes observed for binding the two ligands. To aid interpretation of the calorimetric data, the first crystal structure of a small, guanylyl ribonuclease bound to 3'IMP was determined to 2.0 A resolution. This structure has revealed small yet unexpected changes in the ligand conformation and differences in the conformations of the side-chains contacting the sugar and phosphate moieties as compared to the 3'GMP complex. The structural data suggest the less favorable enthalpy change is due to an overall lengthening of the contacts between RNase Sa and 3'IMP as compared to 3'GMP. The long-range interaction between N2 and Glu41 is critical for positioning of the nucleotide in the binding cleft for optimal contact formation. Thus, combined, the data demonstrate how a long-range interaction can have a significant impact on nucleotide binding affinity and energetics. |
| Databáze: | OpenAIRE |
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