title: The crystal structure of ribonuclease A in complex with thymidine-3'-monophosphate provides further insight into ligand binding.
abstract: Thymidine-3'-monophosphate (3'-TMP) is a competitive inhibitor analogue of the 3'-CMP and 3'-UMP natural product inhibitors of bovine pancreatic ribonuclease A (RNase A). Isothermal titration calorimetry experiments show that 3'-TMP binds the enzyme with a dissociation constant (K(d)) of 15 microM making it one of the strongest binding members of the five natural bases found in nucleic acids (A, C, G, T, and U). To further investigate the molecular properties of this potent natural affinity, we have determined the crystal structure of bovine pancreatic RNase A in complex with 3'-TMP at 1.55 A resolution and we have performed NMR binding experiments with 3'-CMP and 3'-TMP. Our results show that binding of 3'-TMP is very similar to other natural and non-natural pyrimidine ligands, demonstrating that single nucleotide affinity is independent of the presence or absence of a 2'-hydroxyl on the ribose moiety of pyrimidines and suggesting that the pyrimidine binding subsite of RNase A is not a significant contributor of inhibitor discrimination. Accumulating evidence suggests that very subtle structural, chemical, and potentially motional variations contribute to ligand discrimination in this enzyme.
authors.affiliation: Department of Chemistry, Yale University, New Haven, Connecticut 06520, USA.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Doucet
authors.forename: Thusitha B
authors.initials: TB
authors.lastname: Jayasundera
authors.forename: Miljan
authors.initials: M
authors.lastname: Simonović
authors.forename: J Patrick
authors.initials: JP
authors.lastname: Loria
mesh_terms: D000818:Animals; D002417:Cattle; D018360:Crystallography, X-Ray; D003568:Cytidine Monophosphate / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008024:Ligands; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics; D013938:Thymidine Monophosphate / Q000737:chemistry* / Q000378:metabolism
chemical_list: D008024:Ligands; D013938:Thymidine Monophosphate; D012259:Ribonuclease, Pancreatic; D003568:Cytidine Monophosphate