Effect of heterodimer partner RXRα on PPARγ activation function-2 helix in solution
Section snippets
Materials and methods
Materials. The PPARγ ligand GW1929 and RXRα ligand 9cRA were purchased from Sigma–Aldrich (St. Louis, MO). The PPARγ LBD (a.a. Q203–Y477) cDNA was amplified from human adipose cDNA library and inserted at the NdeI/XhoI restriction sites of the pET15b expression vector as described [4]. DNA sequencing analysis confirmed the pET15b-PPARγ LBD sequence. The unlabeled RXRα LBD, PPARγ LBD, and U-[2H, 15N, 13C]-enriched PPARγ LBD were expressed in Escherichia coli BL21-DE3 bacteria, isolated by Talon
Results
The PPARγ LBD was purified as monomer to more than 95% chemical homogeneity by gel filtration chromatography. The dissociation constant Kd of GW1929 binding to the PPARγ LBD was 19 ± 10 nM by fluorescence titration. This value was similar to the reported half-maximal activation EC50 of 9 ± 7 nM for GW1929 [8], suggesting that the purified PPARγ LBD was functional. The gel filtration chromatography of a heterodimer sample and the subsequent SDS–PAGE analysis of the elution fractions showed that the
Discussion
In this study, we have established sequence-specific 13Cα, 13Cβ, and 13CO resonance assignments for over 95% of the 275 residues in the GW1929-bound PPARγ LBD monomer. The chemical shift-derived secondary structure generally agrees with the crystal structure of the PPARγ LBD-GI262570-coactivator peptide ternary complex [10]. Both GW1929 [8] and GI262570 [10] are N-aryl tyrosine derivatives that were designed to mimic the interactions between the PPARγ H3, H5, H10, and AF-2 helices and the
Acknowledgments
This work was supported by grants from the National Institutes of Health (NIH) DK59501 (E. Li), Washington University Digestive Diseases Research Core Center (DK52574), a pilot/feasibility award (J. Lu) from the Washington University Center for Human Nutrition (DK56341). We thank Mr. Weidong Wen for preparing human adipose cDNA library and Mr. Bob McGibbon for assistance in protein purification.
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