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J. Biol. Chem., Vol. 282, Issue 23, 17314-17324, June 8, 2007

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Insights into the Mechanism of Partial Agonism

CRYSTAL STRUCTURES OF THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR LIGAND-BINDING DOMAIN IN THE COMPLEX WITH TWO ENANTIOMERIC LIGANDS^*

Giorgio Pochetti¹², Cristina Godio¹, Nico Mitro, Donatella Caruso, Andrea Galmozzi, Samuele Scurati, Fulvio Loiodice^¶, Giuseppe Fracchiolla^¶, Paolo Tortorella^¶, Antonio Laghezza^¶, Antonio Lavecchia^||, Ettore Novellino^||, Fernando Mazza^**, and Maurizio Crestani³

From the Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Montelibretti, 00016 Monterotondo Stazione, Roma, Italia, Laboratorio "Giovanni Galli" di Biochimica e Biologia Molecolare dei Lipidi e di Spettrometria di Massa, Dipartimento di Scienze Farmacologiche, Università degli Studi di Milano, 20133 Milano, Italia, ^¶Dipartimento Farmaco-Chimico, Università degli Studi di Bari, 70125 Bari, Italia, ^||Dipartimento di Chimica Farmaceutica, Università degli Studi di Napoli, 80131 Napoli, Italia, and ^**Dipartimento di Chimica, Ingegneria Chimica e Materiali, Università di L'Aquila, 67010 L'Aquila, Italia

The peroxisome proliferator-activated receptors (PPARs) are transcriptional regulators of glucose and lipid metabolism. They are activated by natural ligands, such as fatty acids, and are also targets of synthetic antidiabetic and hypolipidemic drugs. By using cell-based reporter assays, we studied the transactivation activity of two enantiomeric ureidofibrate-like derivatives. In particular, we show that the R-enantiomer, (R)-1, is a full agonist of PPAR, whereas the S-enantiomer, (S)-1, is a less potent partial agonist. Most importantly, we report the x-ray crystal structures of the PPAR ligand binding domain complexed with the R- and the S-enantiomer, respectively. The analysis of the two crystal structures shows that the different degree of stabilization of the helix 12 induced by the ligand determines its behavior as full or partial agonist. Another crystal structure of the PPAR·(S)-1 complex, only differing in the soaking time of the ligand, is also presented. The comparison of the two structures of the complexes with the partial agonist reveals significant differences and is suggestive of the possible coexistence in solution of transcriptionally active and inactive forms of helix 12 in the presence of a partial agonist. Mutation analysis confirms the importance of Leu⁴⁶⁵, Leu⁴⁶⁹, and Ile⁴⁷² in the activation by (R)-1 and underscores the key role of Gln²⁸⁶ in the PPAR activity.

Received for publication, March 16, 2007

The atomic coordinates and structure factors (code 2I4J, 2I4P, and 2I4Z) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by Italian Ministry of University and Research Grant Progetti di Ricerca di Interesse Nazionale 2005033023. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-5.

¹ These two authors contributed equally to this work.

² To whom correspondence may be addressed: Istituto di Cristallografia, Sede di Monterotondo, Area della Ricerca ROMA 1, CNR, Via Salaria Km 29,300, 00016 Monterotondo Stazione, Roma, Italia. Tel.: 39-06-90672633; Fax: 39-06-90672630; E-mail: giorgio.pochetti{at}ic.cnr.it.

³ To whom correspondence may be addressed: Laboratorio "Giovanni Galli" di Biochimica e Biologia Molecolare dei Lipidi e di Spettrometria di Massa, Dipartimento di Scienze Farmacologiche, Università degli Studi di Milano, via Balzaretti 9, 20133 Milano, Italia. Tel.: 39-02-50318393/1; Fax: 39-02-50318391; E-mail: Maurizio.Crestani{at}unimi.it.

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