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J. Biol. Chem., Vol. 282, Issue 43, 31373-31379, October 26, 2007

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NMR Solution Structure of Lipocalin-type Prostaglandin D Synthase

EVIDENCE FOR PARTIAL OVERLAPPING OF CATALYTIC POCKET AND RETINOIC ACID-BINDING POCKET WITHIN THE CENTRAL CAVITY^*

Shigeru Shimamoto, Takuya Yoshida, Takashi Inui^¶, Keigo Gohda^||, Yuji Kobayashi^**, Ko Fujimori^¶**, Toshiharu Tsurumura^¶, Kosuke Aritake^¶, Yoshihiro Urade^¶, and Tadayasu Ohkubo¹

From the Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan, ^¶Department of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan, ^||Computer-Aided Molecular Modeling Research Center Kansai, 1-3-12 Honjyo-cho, Higashinada-ku, Kobe, Hyogo 658-0012, Japan, and ^**Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH₂, a common precursor of various prostanoids, to produce PGD₂, an endogenous somnogen and nociceptive modulator, in the brain. L-PGDS is a member of the lipocalin superfamily and binds lipophilic substances, such as retinoids and bile pigments, suggesting that L-PGDS is a dual functional protein acting as a PGD₂-synthesizing enzyme and a transporter for lipophilic ligands. In this study we determined by NMR the three-dimensional structure of recombinant mouse L-PGDS with the catalytic residue Cys-65. The structure of L-PGDS exhibited the typical lipocalin fold, consisting of an eight-stranded, antiparallel -barrel and a long -helix associated with the outer surface of the barrel. The interior of the barrel formed a hydrophobic cavity opening to the upper end of the barrel, the size of which was larger than those of other lipocalins, and the cavity contained two pockets. Molecular docking studies, based on the result of NMR titration experiments with retinoic acid and PGH₂ analog, revealed that PGH₂ almost fully occupied the hydrophilic pocket 1, in which Cys-65 was located and all-trans-retinoic acid occupied the hydrophobic pocket 2, in which amino acid residues important for retinoid binding in other lipocalins were well conserved. Mutational and kinetic studies provide the direct evidence for the PGH₂ binding mode. These results indicated that the two binding sites for PGH₂ and retinoic acid in the large cavity of L-PGDS were responsible for the broad ligand specificity of L-PGDS and the non-competitive inhibition of L-PGDS activity by retinoic acid.

Received for publication, January 5, 2007 , and in revised form, August 10, 2007.

The atomic coordinates and structure factors (code 2E4J) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The chemical shift assignments for free form of L-PGDS have been deposited in the BioMagRes Data Bank (www.bmrb.wisc.edu) under the accession number BMRB 10137.

^* This work was supported in part by of Ministry of Education, Culture, Sports, Science and Technology Grants-in-Aid for Scientific Research 18054021 (to T. O.) and 19590094 (to K. A.). 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 Table S1.

¹ To whom correspondence should be addressed. Tel.: 81-6-6879-8223; Fax: 81-6-6879-8221; E-mail: ohkubo{at}phs.osaka-u.ac.jp.

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