NMR studies of an FK-506 analog, [U-13C]ascomycin, bound to FK-506-binding protein.
Multidimensional, heteronuclear NMR methods were used to determine the complete 1H and 13C resonance assignments for [U-13C]ascomycin bound to recombinant FKBP, including stereospecific assignment of all 22 methylene protons. The conformation of ascomycin was then determined from an analysis of NOEs observed in a 13C-edited 3D HMQC-NOESY spectrum of the [U-13C]ascomycin/FKBP. This structure is found to be quite different from the solution structure of the two forms of uncomplexed FK-506. However, it is very similar to the X-ray crystal structure of FK-506 bound to FKBP, rms deviation = 0.56 A. The methods used for resonance assignment and structure calculation are presented in detail. Furthermore, FKBP/ascomycin NOEs are reported which help define the structure of the ascomycin binding pocket. This structural information obtained in solution was compared to the recently described X-ray crystal structure of the FKBP/FK-506 complex.
Petros
A M
AM
Pharmaceutical Discovery Division, Abbott Park, Illinois 60064.
Gemmecker
G
G
Neri
P
P
Olejniczak
E T
ET
Nettesheim
D
D
Xu
R X
RX
Gubbins
E G
EG
Smith
H
H
Fesik
S W
SW
D002247:Carbon Isotopes; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism; D016559:Tacrolimus / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D022021:Tacrolimus Binding Proteins; D014961:X-Ray Diffraction
D002247:Carbon Isotopes; D002352:Carrier Proteins; D011994:Recombinant Proteins; C058028:immunomycin; D022021:Tacrolimus Binding Proteins; D016559:Tacrolimus