title: An NMR strategy for fragment-based ligand screening utilizing a paramagnetic lanthanide probe.
abstract: A nuclear magnetic resonance-based ligand screening strategy utilizing a paramagnetic lanthanide probe is presented. By fixing a paramagnetic lanthanide ion to a target protein, a pseudo-contact shift (PCS) and a paramagnetic relaxation enhancement (PRE) can be observed for both the target protein and its bound ligand. Based on PRE and PCS information, the bound ligand is then screened from the compound library and the structure of the ligand-protein complex is determined. PRE is an isotropic paramagnetic effect observed within 30 Å from the lanthanide ion, and is utilized for the ligand screening in the present study. PCS is an anisotropic paramagnetic effect providing long-range (~40 Å) distance and angular information on the observed nuclei relative to the paramagnetic lanthanide ion, and utilized for the structure determination of the ligand-protein complex. Since a two-point anchored lanthanide-binding peptide tag is utilized for fixing the lanthanide ion to the target protein, this screening method can be generally applied to non-metal-binding proteins. The usefulness of this strategy was demonstrated in the case of the growth factor receptor-bound protein 2 (Grb2) Src homology 2 (SH2) domain and its low- and high-affinity ligands.
authors.affiliation: Department of Structural Biology, Faculty of Advanced Life Science, Hokkaido University, N-21, W-11, Sapporo 001-0021, Japan.
authors.forename: Tomohide
authors.initials: T
authors.lastname: Saio
authors.forename: Kenji
authors.initials: K
authors.lastname: Ogura
authors.forename: Kazumi
authors.initials: K
authors.lastname: Shimizu
authors.forename: Masashi
authors.initials: M
authors.lastname: Yokochi
authors.forename: Terrence R
authors.initials: TR
authors.lastname: Burke
authors.forename: Fuyuhiko
authors.initials: F
authors.lastname: Inagaki
mesh_terms: D001665:Binding Sites; D051380:GRB2 Adaptor Protein / Q000737:chemistry*; D028581:Lanthanoid Series Elements; D008024:Ligands; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D018909:src Homology Domains
chemical_list: D051380:GRB2 Adaptor Protein; D028581:Lanthanoid Series Elements; D008024:Ligands