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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
Author(s) -
Fejzo Jasna,
Lepre Christopher A.,
Peng Jeffrey W.,
Su Michael S.S.,
Thomson John A.,
Moore Jonathan M.
Publication year - 1996
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560050918
Subject(s) - fkbp , chemistry , calcineurin , heteronuclear single quantum coherence spectroscopy , ligand (biochemistry) , protein subunit , nuclear magnetic resonance spectroscopy , stereochemistry , protein quaternary structure , receptor , biochemistry , transplantation , medicine , surgery , gene
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)S. Analysis of 1 H‐ 13 C HSQC data acquired for the FKBP‐12/ 13 C‐FK506 and FKBP‐12/ 13 C‐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‐CH 2 , 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‐CH 2 protons. The tri‐substituted olefin and cyclohexyl groups also show moderate broadening at the 27‐Me, 28‐CH, and 30‐CH 2 positions, respectively. Unexpectedly, little line broadening was observed for the allyl resonances of FK506 in the quaternary complex, although 13 C longitudinal relaxation measurements suggest this group also makes contacts with calcineurin. In addition, intermolecular transfer NOE peaks were observed for the allyl 37‐CH 2 , 21‐CH, 30‐CH 2 , 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.