title: Investigating protein-ligand interactions with a mutant FKBP possessing a designed specificity pocket.
abstract: Using structure-based design and protein mutagenesis we have remodeled the FKBP12 ligand binding site to include a sizable, hydrophobic specificity pocket. This mutant (F36V-FKBP) is capable of binding, with low or subnanomolar affinities, novel synthetic ligands possessing designed substituents that sterically prevent binding to the wild-type protein. Using binding and structural analysis of bumped compounds, we show here that the pocket is highly promiscuous-capable of binding a range of hydrophobic alkyl and aryl moieties with comparable affinity. Ligand affinity therefore appears largely insensitive to the degree of occupancy or quality of packing of the pocket. NMR spectroscopic analysis indicates that similar ligands can adopt radically different binding modes, thus complicating the interpretation of structure-activity relationships.
authors.affiliation: ARIAD Gene Therapeutics, 26 Landsdowne Street, Cambridge, Massachusetts 02139-4234, USA.
authors.forename: W
authors.initials: W
authors.lastname: Yang
authors.forename: L W
authors.initials: LW
authors.lastname: Rozamus
authors.forename: S
authors.initials: S
authors.lastname: Narula
authors.forename: C T
authors.initials: CT
authors.lastname: Rollins
authors.forename: R
authors.initials: R
authors.lastname: Yuan
authors.forename: L J
authors.initials: LJ
authors.lastname: Andrade
authors.forename: M K
authors.initials: MK
authors.lastname: Ram
authors.forename: T B
authors.initials: TB
authors.lastname: Phillips
authors.forename: M R
authors.initials: MR
authors.lastname: van Schravendijk
authors.forename: D
authors.initials: D
authors.lastname: Dalgarno
authors.forename: T
authors.initials: T
authors.lastname: Clackson
authors.forename: D A
authors.initials: DA
authors.lastname: Holt
mesh_terms: D000081:Acetamides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D001555:Benzene Derivatives / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D020104:Immunophilins / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation; D015202:Protein Engineering; D013329:Structure-Activity Relationship; D022021:Tacrolimus Binding Proteins
chemical_list: D000081:Acetamides; D001555:Benzene Derivatives; D008024:Ligands; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins