Dynamic NMR studies of ligand-receptor interactions: design and analysis of a rapidly exchanging complex of FKBP-12/FK506 with a 24 kDa calcineurin fragment.
Dynamic NMR methods, such as differential line broadening and transferred NOE spectroscopy, are normally reserved for the study of small molecule ligand interactions with large protein receptors. Using a combination of isotope labeling and isotope edited NMR, we have extended these techniques to characterize interactions of a much larger protein/drug complex, FKBP-12/ FK506 with its receptor protein, calcineurin. In order to examine this multicomponent system by dynamic NMR methods, the 93 kDa, tightly bound FKBP-12/FK506/Cn complex was replaced with a lower affinity, rapidly exchanging system consisting of FKBP-12/FK506 (13 kDa), recombinant calcineurin subunit B (CnB) (20 kDa), and a synthetic peptide (4 kDa) corresponding to the B binding domain (BBD) of calcineurin catalytic subunit A (CnA). Analysis of 1H-13C HSQC data acquired for the FKBP-12/ 13C-FK506 and FKBP-12/13C-FK506/CnB/BBD complexes indicates that FKBP-12/FK506 and CnB/BBD are in fast exchange in the quaternary complex. Comparison of proton line widths shows significant broadening of resonances along the macrocycle backbone at 13-CH, 13-OMe, 15-OMe, 18-CH2, 20-CH, 21-CH, and 25-Me, as well as moderate broadening on the macrocycle backbone at 17-Me, 24-CH, and the pyranose 12-CH2 protons. The tri-substituted olefin and cyclohexyl groups also show moderate broadening at the 27-Me, 28-CH, and 30-CH2 positions, respectively. Unexpectedly, little line broadening was observed for the allyl resonances of FK506 in the quaternary complex, although 13C longitudinal relaxation measurements suggest this group also makes contacts with calcineurin. In addition, intermolecular transfer NOE peaks were observed for the allyl 37-CH2, 21-CH, 30-CH2, 13-OMe, 15-OMe, 17-Me, 25-Me, and 27-Me groups, indicating that these are potential sites on the FK506 molecule that interact with calcineurin.
Fejzo
J
J
Vertex Pharmaceuticals Incorporated, Cambridge, Massachusetts 02139-4242, USA.
Lepre
C A
CA
Peng
J W
JW
Su
M S
MS
Thomson
J A
JA
Moore
J M
JM
D000595:Amino Acid Sequence; D001665:Binding Sites; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins; D022021:Tacrolimus Binding Proteins
D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D022021:Tacrolimus Binding Proteins