title: Small-molecule binding sites on proteins established by paramagnetic NMR spectroscopy.
abstract: Determining the three-dimensional structure of a small molecule-protein complex with weak affinity can be a significant challenge. We present a paramagnetic NMR method to determine intermolecular structure restraints based on pseudocontact shifts (PCSs). Since the ligand must be in fast exchange between free and bound states and the fraction bound can be as low as a few percent, the method is ideal for ligands with high micromolar to millimolar dissociation constants. Paramagnetic tags are attached, one at a time, in a well-defined way via two arms at several sites on the protein surface. The ligand PCSs were measured from simple 1D (1)H spectra and used as docking restraints. An independent confirmation of the complex structure was carried out using intermolecular NOEs. The results show that structures derived from these two approaches are similar. The best results are obtained if the magnetic susceptibility tensors of the tags are known, but it is demonstrated that with two-armed probes, the magnetic susceptibility tensor can be predicted with sufficient accuracy to provide a low-resolution model of the ligand orientation and the location of the binding site in the absence of isotope-labeled protein. This approach can facilitate fragment-based drug discovery in obtaining structural information on the initial fragment hits.
authors.affiliation: Gorlaeus Laboratories, Leiden Institute of Chemistry, Leiden University, Post Office Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Jia-Ying
authors.initials: JY
authors.lastname: Guan
authors.forename: Peter H J
authors.initials: PH
authors.lastname: Keizers
authors.forename: Wei-Min
authors.initials: WM
authors.lastname: Liu
authors.forename: Frank
authors.initials: F
authors.lastname: Löhr
authors.forename: Simon P
authors.initials: SP
authors.lastname: Skinner
authors.forename: Edwin A
authors.initials: EA
authors.lastname: Heeneman
authors.forename: Harald
authors.initials: H
authors.lastname: Schwalbe
authors.forename: Marcellus
authors.initials: M
authors.lastname: Ubbink
authors.forename: Gregg
authors.initials: G
authors.lastname: Siegal
mesh_terms: D001665:Binding Sites; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D022061:Tacrolimus Binding Protein 1A / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D008024:Ligands; D022061:Tacrolimus Binding Protein 1A