STRUCTURAL ANALYSIS OF RAPAMYCIN'S ROLE IN BINDING FKBP12 AND FRAP. Jon Clardy*, Jungwon Choi*, Jie Chen[[daggerdbl]], and Stuart L. Schreiber[[daggerdbl]], *Department of Chemistry, Cornell University, Ithaca, NY 14853-1301. [[daggerdbl]]Howard Hughes Medical Institute, Department of Chemistry, Harvard University, Cambridge, MA 02138, USA
The immunosuppressive drug rapamycin works by binding together two proteins: the FK506 binding protein (FKBP12) and the FKBP-rapamycin associated protein (FRAP). The crystal structure of the immunosuppressive agent rapamycin complexed with human FK506 binding protein (FKBP12) and the FKBP12-rapamycin binding domain (FRB) of FKBP12-rapamycin associated protein (FRAP), reveals two proteins bound together through rapamycin's ability to simultaneously occupy two different hydrophobic binding pockets. FKBP12 has a hydrophobic binding pocket between a large ß-sheet and a short amphipathic [[alpha]]-helix. FRB is a four-helix bundle, and the crossing region between two antiparallel helices forms the rapamycin binding pocket. The structure shows extensive interactions between rapamycin and both proteins but only modest protein-protein interactions. The structure also provides, among other things, an understanding of the critical nature of the serine 2,035 residue, and the first structural information about the growing family of proteins related to the ataxia telangicetasia mutant (ATM) gene product.
Crystals of the triple complex form in space group P212121 with a = 44.63, b = 52.14, and c = 102.53 Å and one FKBP12-rapamycin-FRB complex in the asymmetric unit. Data were collected to 2.7 Å with a rotating anode source. A phasing model was found using MR (FKBP12) and SIRAS (HgCl2 derivative). The current model has R = 19.3% (Rfree = 29.9%) for the 6206 reflections between 8 and 2.7 Å and good geometry (r.m.s. deviations of 0.008Å for bond lengths and 1.48[[ring]] for bond angles) for a model with 1639 protein atoms, 65 rapamycin atoms, and 23 solvent atoms--not counting hydrogens.