title: Solution structure of FK506 bound to the R42K, H87V double mutant of FKBP-12.
abstract: The binding of the FK506/FKBP-12 complex to calcineurin (CN), its putative target for immunosuppression, involves recognition of solvent-exposed regions of the ligand as well as FKBP-12 residues near the active site. The R42K, H87V double mutation of FKBP-12 decreases the CN affinity of the complex by 550-fold [Aldape, R. A., Futer, O., DeCenzo, M. T., Jarrett, B. P., Murcko, M. A., & Livingston, D. J. (1992) J. Biol. Chem. 267, 16029-16032]. This work reports the solution structure of 13C-labeled FK506 bound to R42K, H87V FKBP-12. Assignments and NOE measurements at three mixing times were made from inverse-detected 1H-13C NMR experiments. Structures were calculated by several different methods, including distance geometry, restrained molecular dynamics, and molecular dynamics with time-averaged restraints. The NMR structures of the ligand are very well defined by the NOE restraints and differ slightly from the X-ray structure in regions that are involved in crystal packing. Comparison with the NMR structure of FK506 bound to wild-type FKBP-12 reveals that the R42K, H87V mutation causes the ligand backbone near C16 to move by 2.5 to 4.5 A, reorients 15-MeO by 90 degrees, and shifts 13-MeO by approximately 1.5 A. FK506 appears to undergo a concerted, mutationally induced shift in the binding pocket, with the greatest changes occurring in the effector region of the drug. The altered effector conformation of mutant-bound FK506 may perturb interactions between the drug and CN, thus accounting for the effect of the double mutation upon the CN inhibitory activity of the complex.
authors.affiliation: Vertex Pharmaceuticals Incorporated, Cambridge, Massachusetts 02139-4211.
authors.forename: C A
authors.initials: CA
authors.lastname: Lepre
authors.forename: D A
authors.initials: DA
authors.lastname: Pearlman
authors.forename: J W
authors.initials: JW
authors.lastname: Cheng
authors.forename: M T
authors.initials: MT
authors.lastname: DeCenzo
authors.forename: D J
authors.initials: DJ
authors.lastname: Livingston
authors.forename: J M
authors.initials: JM
authors.lastname: Moore
mesh_terms: D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000037:antagonists & inhibitors; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003196:Computer Graphics; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins; D012996:Solutions; D016559:Tacrolimus / Q000737:chemistry* / Q000378:metabolism; D022021:Tacrolimus Binding Proteins
chemical_list: D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D012996:Solutions; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D022021:Tacrolimus Binding Proteins; D016559:Tacrolimus