title: Probing hot spots at protein-ligand binding sites: a fragment-based approach using biophysical methods.
abstract: Mapping interactions at protein-ligand binding sites is an important aspect of understanding many biological reactions and a key part of drug design. In this paper, we have used a fragment-based approach to probe "hot spots" at the cofactor-binding site of a model dehydrogenase, Escherichia coli ketopantoate reductase. Our strategy involved the breaking down of NADPH (Kd = 300 nM) into smaller fragments and the biophysical characterization of their binding using WaterLOGSY NMR spectroscopy, isothermal titration calorimetry (ITC), and inhibition studies. The weak binding affinities of fragments were measured by direct ITC titrations under low c value conditions. The 2'-phosphate and the reduced nicotinamide groups were found to contribute a large part of the binding energy. A combination of ITC and site-directed mutagenesis enabled us to locate the fragments at separate hot spots on opposite ends of the cofactor-binding site. This study has identified structural determinants for cofactor recognition that represent a blueprint for future inhibitor design.
authors.affiliation: University Chemical Laboratory, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
authors.forename: Alessio
authors.initials: A
authors.lastname: Ciulli
authors.forename: Glyn
authors.initials: G
authors.lastname: Williams
authors.forename: Alison G
authors.initials: AG
authors.lastname: Smith
authors.forename: Tom L
authors.initials: TL
authors.lastname: Blundell
authors.forename: Chris
authors.initials: C
authors.lastname: Abell
mesh_terms: D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D002151:Calorimetry; D004926:Escherichia coli / Q000201:enzymology; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D008024:Ligands; D009711:Nucleotides; D000429:Alcohol Oxidoreductases; C021934:2-dehydropantoate 2-reductase