title: Rapid identification of ligand-binding sites by using an assignment-free NMR approach.
abstract: In this study, we developed an assignment-free approach for rapid identification of ligand-binding sites in target proteins by using NMR. With a sophisticated cell-free stable isotope-labeling procedure that introduces (15)N- or (13)C-labels to specific atoms of target proteins, this approach requires only a single series of ligand titrations with labeled targets. Using titration data, ligand-binding sites in the target protein can be identified without time-consuming assignment procedures. We demonstrated the feasibility of this approach by using structurally well-characterized interactions between mitogen-activated protein (MAP) kinase p38α and its inhibitor 2-amino-3-benzyloxypyridine. Furthermore, we confirmed the recently proposed fatty acid binding to p38α and confirmed the fatty acid-binding site in the MAP kinase insert region.
authors.affiliation: Japan Biological Informatics Consortium (JBIC) , 2-3-26 Aomi, Koto-ku, Tokyo 135-0064, Japan.
authors.forename: Yuya
authors.initials: Y
authors.lastname: Kodama
authors.forename: Koh
authors.initials: K
authors.lastname: Takeuchi
authors.forename: Nobuhisa
authors.initials: N
authors.lastname: Shimba
authors.forename: Kohki
authors.initials: K
authors.lastname: Ishikawa
authors.forename: Ei-ichiro
authors.initials: E
authors.lastname: Suzuki
authors.forename: Ichio
authors.initials: I
authors.lastname: Shimada
authors.forename: Hideo
authors.initials: H
authors.lastname: Takahashi
mesh_terms: D001665:Binding Sites; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005227:Fatty Acids / Q000378:metabolism; D005240:Feasibility Studies; D057166:High-Throughput Screening Assays; D006801:Humans; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D048308:Mitogen-Activated Protein Kinase 14 / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D011725:Pyridines / Q000737:chemistry / Q000494:pharmacology; D013997:Time Factors
chemical_list: D005227:Fatty Acids; D008024:Ligands; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D048308:Mitogen-Activated Protein Kinase 14; C023666:pyridine