Crystal Structure of the Ligand Binding Domain of the Human Nuclear Receptor PPARγ

Abstract

The peroxisome proliferator-activated receptors (PPAR) are members of the nuclear receptor supergene family and are considered as key sensors of both lipid and glucose homeostasis. The role of the PPAR␥ isoform in glucose metabolism is illustrated by the fact that antidiabetic thiazolidinediones have been shown to be bona fide PPAR␥ ligands. Here we report the crystal structure of apo-PPAR␥ ligand binding domain (LBD) determined to 2.9-Å resolution. Although the structure of apo-PPAR␥-LBD retains the overall fold described previously for other nuclear receptor LBDs, three distinct structural differences are evident. 1) The core AF-2 activation domain of apo-PPAR␥ LBD is folded back toward the predicted ligand binding pocket similar to that observed in the holo-forms of other nuclear receptors. 2) The proposed ligand binding pocket of apo-PPAR␥-LBD is larger and more accessible to the surface in contrast to other LBDs. 3) The region of the LBD called the -loop is extended in PPAR␥ and contains additional structural elements. Taken together, the apo-PPAR␥-LBD structure is in several aspects different from previously described LBDs. Given the central role of PPAR␥ as a mediator in glucose regulation, the structure should be an important tool in the development of improved anti-diabetic agents.

A New Class of Peroxisome Proliferator-activated Receptor Agonists with a Novel Binding Epitope Shows Antidiabetic Effects

“…Resolutions of the complexes were 2.8, 3.15, and 2.9 Å, respectively ( Table I). The overall fold of the PPAR␥-LBD shows only minor differences as compared with the previously determined apoPPAR␥ structure (26). The major differences are in the region connecting helix 2Ј and helix 3, a region with high mobility.…”

“…Furthermore, the 2-BABAs are situated at the entrance of the active site and do not interact directly with helix 12. There are three unrelated crystallographic apo structures of PPAR␥ (21,26), of which two closely resemble the agonistic structures in terms of helix 12 position and interactions, thus giving no hints of conformational changes related to ligand activation. In the third structure helix 12 adopts a different conformation, slightly protruding away from the LBD, and could therefore more easily be interpreted as an inactive state.…”

“…Expression and Isolation of hPPAR␥-LBD-A construct described previously and used for the crystallization of the apoPPAR␥-LBD (26) was redesigned by site-directed mutagenesis to harbor a factor X a cleavage site, resulting in the deduced amino acid sequence MGHHHHHHSGSGTIEGR(Leu 204 -Tyr 477 ). The expression and isolation protocol was performed as described previously (26), except for the addition of an ion exchange (Resource Q, pH 8.0) and a molecular sieving step (Superdex 75) after the first crude immobilized metal ion affinity chromatography step.…”

“…The expression and isolation protocol was performed as described previously (26), except for the addition of an ion exchange (Resource Q, pH 8.0) and a molecular sieving step (Superdex 75) after the first crude immobilized metal ion affinity chromatography step. Isolated PPAR␥-LBD was dialyzed against 20 mM Tris/HCl, pH 8.0, and concentrated to 9 mg/ml.…”

“…Structural Analysis of Cocrystal Complexes-Initial docking studies of the 2-BABA scaffold to the apoPPAR␥-LBD structure (26) in combination with the bioisosteric properties of the TZD headgroup and carboxylate group of the 2-BABA compounds suggested an anchoring mode similar to that of eicosapentaenoic acid to PPAR␦ (5), with the carboxylic headgroup interacting with Tyr-473 in helix 12. To further elucidate this hypothesis, the crystal structures of BVT.13, BVT.762, and BVT.763 in complex with PPAR␥-LBD and a GRIP-1-derived coactivator peptide were solved by molecular replacement.…”