Structural determinants of ligand binding selectivity between the peroxisome proliferator-activated receptors.

The peroxisome proliferator-activated receptors (PPARs) are transcriptional regulators of glucose, lipid, and cholesterol metabolism. We report the x-ray crystal structure of the ligand binding domain of PPAR alpha (NR1C1) as a complex with the agonist ligand GW409544 and a coactivator motif from the steroid receptor coactivator 1. Through comparison of the crystal structures of the ligand binding domains of the three human PPARs, we have identified molecular determinants of subtype selectivity. A single amino acid, which is tyrosine in PPAR alpha and histidine in PPAR gamma, imparts subtype selectivity for both thiazolidinedione and nonthiazolidinedione ligands. The availability of high-resolution cocrystal structures of the three PPAR subtypes will aid the design of drugs for the treatments of metabolic and cardiovascular diseases.

Structural basis for autorepression of retinoid X receptor by tetramer formation and the AF-2 helix.

The 9-cis-retinoic acid receptors (RXRalpha, RXRbeta, and RXRgamma) are nuclear receptors that play key roles in multiple hormone-signaling pathways. Biochemical data indicate that, in the absence of ligand, RXR can exist as an inactive tetramer and that its dissociation, induced by ligand, is important for receptor activation. In this article we report the inactivated tetramer structures of the RXRalpha ligand-binding domain (LBD), either in the absence of or in the presence of a nonactivating ligand. These structures reveal that the RXR LBD tetramer forms a compact, disc-shaped complex, consisting of two symmetric dimers that are packed along helices 3 and 11. In each monomer, the AF-2 helix protrudes away from the core domain and spans into the coactivator binding site in the adjacent monomer of the symmetric dimer. In this configuration, the AF-2 helix physically excludes the binding of coactivators and suggests an autorepression mechanism that is mediated by the AF-2 helix within the tetramer. The RXR-tetramer interface is assembled from amino acids that are conserved across several closely related receptors, including the HNF4s and COUP transcription factors, and may therefore provide a model for understanding structure and regulation of this subfamily of nuclear receptors.

Asymmetry in the PPARgamma/RXRalpha crystal structure reveals the molecular basis of heterodimerization among nuclear receptors.

The nuclear receptor PPARgamma/RXRalpha heterodimer regulates glucose and lipid homeostasis and is the target for the antidiabetic drugs GI262570 and the thiazolidinediones (TZDs). We report the crystal structures of the PPARgamma and RXRalpha LBDs complexed to the RXR ligand 9-cis-retinoic acid (9cRA), the PPARgamma agonist rosiglitazone or GI262570, and coactivator peptides. The PPARgamma/RXRalpha heterodimer is asymmetric, with each LBD deviated approximately 10 degrees from the C2 symmetry, allowing the PPARgamma AF-2 helix to interact with helices 7 and 10 of RXRalpha. The heterodimer interface is composed of conserved motifs in PPARgamma and RXRalpha that form a coiled coil along helix 10 with additional charge interactions from helices 7 and 9. The structures provide a molecular understanding of the ability of RXR to heterodimerize with many nuclear receptors and of the permissive activation of the PPARgamma/RXRbeta heterodimer by 9cRA.

Alteration of a single amino acid in peroxisome proliferator-activated receptor-alpha (PPAR alpha) generates a PPAR delta phenotype.

Three pharmacologically important nuclear receptors, the peroxisome proliferator-activated receptors (PPARs alpha, gamma, and delta), mediate key transcriptional responses involved in lipid homeostasis. The PPAR alpha and gamma subtypes are well conserved from Xenopus to man, but the beta/delta subtypes display substantial species variations in both structure and ligand activation profiles. Characterization of the avian cognates revealed a close relationship between chick (c) alpha and gamma subtypes to their mammalian counterparts, whereas the third chicken subtype was intermediate to Xenopus (x) beta and mammalian delta, establishing that beta and delta are orthologs. Like xPPAR beta, cPPAR beta responded efficiently to hypolipidemic compounds that fail to activate the human counterpart. This provided the opportunity to address the pharmacological problem as to how drug selectivity is achieved and the more global evolutionary question as to the minimal changes needed to generate a new class of receptor. X-ray crystallography and chimeric analyses combined with site-directed mutagenesis of avian and mammalian cognates revealed that a Met to Val change at residue 417 was sufficient to switch the human and chick phenotype. These results establish that the genetic drive to evolve a novel and functionally selectable receptor can be modulated by a single amino acid change and suggest how nuclear receptors can accommodate natural variation in species physiology.

Molecular recognition of fatty acids by peroxisome proliferator-activated receptors.

The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors for fatty acids (FAs) that regulate glucose and lipid homeostasis. We report the crystal structure of the PPAR delta ligand-binding domain (LBD) bound to either the FA eicosapentaenoic acid (EPA) or the synthetic fibrate GW2433. The carboxylic acids of EPA and GW2433 interact directly with the activation function 2 (AF-2) helix. The hydrophobic tail of EPA adopts two distinct conformations within the large hydrophobic cavity. GW2433 occupies essentially the same space as EPA bound in both conformations. These structures provide molecular insight into the propensity for PPARs to interact with a variety of synthetic and natural compounds, including FAs that vary in both chain length and degree of saturation.

Crystal structure of the catalytic domain of the human cell cycle control phosphatase, Cdc25A.

Cdc25 phosphatases activate the cell division kinases throughout the cell cycle. The 2.3 A structure of the human Cdc25A catalytic domain reveals a small alpha/beta domain with a fold unlike previously described phosphatase structures but identical to rhodanese, a sulfur-transfer protein. Only the active-site loop, containing the Cys-(X)5-Arg motif, shows similarity to the tyrosine phosphatases. In some crystals, the catalytic Cys-430 forms a disulfide bond with the invariant Cys-384, suggesting that Cdc25 may be self-inhibited during oxidative stress. Asp-383, previously proposed to be the general acid, instead serves a structural role, forming a conserved buried salt-bridge. We propose that Glu-431 may act as a general acid. Structure-based alignments suggest that the noncatalytic domain of the MAP kinase phosphatases will share this topology, as will ACR2, a eukaryotic arsenical resistance protein.