Structural and Biochemical Basis for the Binding Selectivity of Peroxisome Proliferator-activated Receptor γ to PGC-1α

Yong Li,Amanda Kovach,Kelly Suino-Powell,Dariusz Martynowski,H Eric Xu

doi:10.1074/jbc.m802040200

Yong Li, Amanda Kovach + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m802040200

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Abstract

The functional interaction between the peroxisome proliferator-activated receptor gamma (PPARgamma) and its coactivator PGC-1alpha is crucial for the normal physiology of PPARgamma and its pharmacological response to antidiabetic treatment with rosiglitazone. Here we report the crystal structure of the PPARgamma ligand-binding domain bound to rosiglitazone and to a large PGC-1alpha fragment that contains two LXXLL-related motifs. The structure reveals critical contacts mediated through the first LXXLL motif of PGC-1alpha and the PPARgamma coactivator binding site. Through a combination of biochemical and structural studies, we demonstrate that the first LXXLL motif is the most potent among all nuclear receptor coactivator motifs tested, and only this motif of the two LXXLL-related motifs in PGC-1alpha is capable of binding to PPARgamma. Our studies reveal that the strong interaction of PGC-1alpha and PPARgamma is mediated through both hydrophobic and specific polar interactions. Mutations within the context of the full-length PGC-1alpha indicate that the first PGC-1alpha motif is necessary and sufficient for PGC-1alpha to coactivate PPARgamma in the presence or absence of rosiglitazone. These results provide a molecular basis for specific recruitment and functional interplay between PPARgamma and PGC-1alpha in glucose homeostasis and adipocyte differentiation.

Highlights

Pocyte differentiation, glucose homeostasis, and inflammatory responses [1, 2]
Upon interaction between the metabolism [30]. These results demonstrate that roscoactivator peptides and the PPAR␥ ligand-binding domain (LBD), excitation with a iglitazone promotes the interaction of coactivator motifs with laser beam at 680 nm causes the donor beads to emit single PPAR␥ and that PGC-1␣ ID1 preferentially binds to PPAR␥
Ser142 and Lys145 of PGC-1␣ contribute to its ability to coactivate PPAR␥. These results reveal that the strong interaction of PGC-1␣ with PPAR␥ is due to hydrophobic binding of its ID1 LXXLL motif and to specific interactions between the two molecules

Summary

EXPERIMENTAL PROCEDURES

Protein Preparation—Both the human PPAR␥ LBD (residues 206 – 477) and the human PGC-1␣ fragment (residues 101– 220), each containing a His tag, were expressed from the expression vector pETDuet (Novagen). The column was washed with extract buffer, and the protein was eluted with a 300-ml gradient to the buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol, and 500 mM imidazole) Both human PPAR␥ LBD and the PGC-1␣ fragment were further purified on a Q-Sepharose column. The relative binding affinity of peptide LXXLL motifs was determined using unlabeled peptides at 500 nM to compete with the binding of biotinylated SRC2-3 to PPAR␥ LBD. To determine which coactivators are preferentially recruited, we performed a peptide profiling experiment using a panel of unlabeled peptides to compete off the binding of the biotinylated third LXXLL motif of SRC2 (SRC2-3) to the rosiglitazone-bound PPAR␥ LBD (Fig. 1B). In the absence of any competing peptides, interaction between the biotinylated SRC2-3 motif and the PPAR␥ LBD yielded a count of 27,000 photons (Fig. 1B)

RESULTS

The overall structure of the

Unique reflections

To assess the importance of these

Findings

DISCUSSION