Spectroscopic analyses of the binding kinetics of 15d-PGJ2 to the PPARgamma ligand-binding domain by multi-wavelength global fitting.

PPARgamma (peroxisome proliferator-activated receptor gamma) is a nuclear receptor that is activated by natural lipid metabolites, including 15d-PGJ2 (15-deoxy-Delta(12,14)-prostaglandin J2). We previously reported that several oxidized lipid metabolites covalently bind to PPARgamma through a Michael-addition to activate transcription. To separate the ligand-entering (dock) and covalent-binding (lock) steps in PPARgamma activation, we investigated the binding kinetics of 15d-PGJ2 to the PPARgamma LBD (ligand-binding domain) by stopped-flow spectroscopy. We analysed the spectral changes of 15d-PGJ2 by multi-wavelength global fitting based on a two-step chemical reaction model, in which an intermediate state represents the 15d-PGJ2-PPARgamma complex without covalent binding. The extracted spectrum of the intermediate state in wild-type PPARgamma was quite similar to the observed spectrum of 15d-PGJ2 in the C285S mutant, which cannot be activated by 15d-PGJ2, indicating that the complex remains in the inactive, intermediate state in the mutant. Thus 'lock' rather than 'dock' is one of the critical steps in PPARgamma activation by 15d-PGJ2.