Flexible Structure of Prostaglandin D Synthase (PGDS) with Various Functions Supporting Sleep Induction, and Maturation and Maintenance of Reproductive System (Press Release)

Release Date: 10 Jul, 2009

BL45XU (RIKEN Structural Biology I)

A research group consisting of scientists from RIKEN and Osaka Bioscience Institute determined the two different steric structures of lipocalin-type prostaglandin D synthase (lipocalin-type PGDS) that catalyzes the synthesis of a sleep-inducing substance.

Flexible change in pouchlike structure enables expression of various functions such as synthesis of sleep-inducing substance.

RIKEN
Osaka Bioscience Institute

Key research achievements
• Discovery of the PGDS pouchlike structure with a lid for opening/closing the structure
• Possibility of inducing brain protection function by combining the flexibility of changing the PGDS structure with the features of substances responsible for Alzheimer's disease
• Relationships between PGDS and diseases, such as obesity due to overeating and arteriosclerosis, remain to be clarified in future research

A research group consisting of scientists from RIKEN (Ryoji Noyori, President) and Osaka Bioscience Institute (Osamu Hayaishi, Chairman of the Board of Trustees) determined the two different steric structures of lipocalin-type prostaglandin D synthase (lipocalin-type PGDS)^*1 that catalyzes the synthesis of a sleep-inducing substance. It was clarified that the synthase has a pouchlike structure with a lid for opening/closing the structure; by flexibly changing its structure, lipocalin-type PGDS supports vital activities, such as sleeping, and maturation. This was accomplished by a joint research team consisting of Masashi Miyano, chief scientist, and Hideo Ago, senior research scientist, of the Structural Biophysics Laboratory, RIKEN SPring-8 Center, and Yoshihiro Urade, head, and Kousuke Aritake, technician, of the Osaka Bioscience Institute.

Lipocalin family proteins^*2 hold physiological substances inside a pouchlike structure. Among them, only PGDS has enzyme activity and catalyzes the synthesis of prostaglandin D₂ (PGD₂), which naturally induces non-rapid-eye-movement (non-REM) sleep.^*3 Two types of PGDS, namely, hematopoietic and lipocalin, are known. Hematopoietic-type PGDS is considered to be associated mainly with immunological and irritation controls. Hematopoietic-type PGDS was discovered in 1997, and the research group clarified its crystalline structure and reaction mechanism around the same time. It was found that hematopoietic-type PGDS is related to myonecrosis, and the research and development of it as a possible drug for treating Duchenne muscular dystrophy^*4 are under way. Another type of PGDS, i.e., lipocalin-type PGDS, is a small protein composed of only 150 amino acids. It functions in various activities, such as sleeping, and maturation, thus supporting our lives. Although the gene sequence of the protein was identified relatively early, it is difficult to produce large quantities of the protein in its active form because its early-stage recombinant form is unstable.

In this study, two types of lipocalin-type PGDS crystals were analyzed using RIKEN Structural Biology I beamline BL45XU at SPring-8, and the scientists involved succeeded in the determination of their steric structures. The result indicates that lipocalin-type PGDS has a pouchlike structure with a lid composed of proteins. Furthermore, taking into account the past reports on the structure of lipocalin-type PGDS such as the results of nuclear magnetic resonance (NMR) analysis^*⁵ conducted by Osaka Bioscience Institute in cooperation with other institutions, they concluded that lipocalin-type PGDS has a very flexible structure. Lipocalin-type PGDS is considered to not only serve as an enzyme protein that catalyzes the synthesis of PGD₂, but also serve as a protein that strongly binds to retinoic acid (a derivative of vitamin A), thyroxin hormone, complex sugars, and even β amyloid (a causative substance of Alzheimer's disease) owing to its flexible structure. Their achievement is expected to accelerate the clarification of the brain protection mechanism and the development of drugs for arteriosclerosis.

The achievement will be published in the Journal of Biological Chemistry in the US on August 14, 2009.

Publication:
"Structural Basis of the Catalytic Mechanism Operating in Open-Closed Conformers of Lipocalin Type Prostaglandin D Synthase"
Takashi Kumasaka, Kosuke Aritake, Hideo Ago, Daisuke Irikura, Toshiharu Tsurumura, Masaki Yamamoto, Masashi Miyano, Yoshihiro Urade, and Osamu Hayaishi
Journal of Biological Chemistry 284 (33), 22344-22352 (2009), published online June 22, 2009.

<Figure>

Fig. 1 Steric structures of lipocalin-type and hematopoietic-type PGDSs

Lipocalin-type PGDS is smaller than hematopoietic-type PGDS. In Lipocalin-type PGDS, the number of spiral α helix structures (spiral structures in the figure) is small and β sheets (indicated by arrows) form the pouchlike structures, where PGD₂ is synthesized (left figure). In contrast, in the case of hematopoietic-type PGDS, PGD₂ is synthesized in a gap where the large area consisting of α helix binds to catalytic glutathione (a black bar with red, green, and blue balls) at the lower part of the PGDS structure, as shown in the right figure.

Fig. 2 Structure of lipocalin-type PGDS with open (B) and closed (C) lids of pouchlike structure

Fig. 2 Structure of lipocalin-type PGDS with open (B) and closed (C) lids of pouchlike structure
A.	Cross-sectional view of superimposed image of open and closed states. (Pink and green indicate the open and closed states, respectively).
B.	Open condition: A pink arrow indicates the open state.
C.	Closed condition: A green arrow indicates the closed state.
D.	Enlarged view of the loop area related to gating of the pouchlike structure. The gating is induced by changes in the structure of the loop. It is considered that an open pouchlike structure (pink double-headed arrows) is closed (green double-headed arrows) by the swinging of the loop (blue arrow).

Fig. 3 Superimposition of lipocalin-type PGDS structures

Image obtained from structural analysis of open and closed states by X-ray analysis (pink indicates open state, and blue indicates closed state), onto which images of 15 typical structures (fine ribbons) of lipocalin-type PGDS determined by NMR analysis (PDB code 2E4J) are superimposed.

Fig. 4 Steric structure onto which amino acid residues at the active sites of lipocalin-type PGDS and hematopoietic-type PGDS are superimposed

Fig. 4 Steric structure onto which amino acid residues at the active sites of lipocalin-type PGDS and hematopoietic-type PGDS are superimposed (You can see one steric structure, when your right and left eyes look at the right and left figures, respectively; focus on the background of the figure, not on the figure itself.) The pink amino acid chain is lipocalin-type PGDS, whereas the light-blue chain is hematopoietic-type PGDS.
Pink background:	catalyst residue and activated amino acid residue.
Green background:	ω-chain bonding moiety in two PGD₂ legs, the fat content of which is higher than that in the other leg.
Yellow background:	positively charged amino acid residue where the bonding moiety at the root of the substrate α chain interacts with the carboxylic terminus.
Blue background:	amino acid residue in which the negatively charged α chain terminus, in the other leg of PGD₂, is stabilized by the positive charge.

<Glossary>

*1 Lipocalin-type prostaglandin D synthase (lipocalin-type PGDS)
Lipocalin-type PGDS belongs to the lipocalin family of proteins, and is the only protein in the family that has enzyme activity.

*2 Lipocalin family proteins
Lipocalin family proteins are capable of holding high-fat physiological substances, such as fatty acids and retinoic acid, in their pouchlike structure, and thus carrying these substances.

*3 Non-rapid-eye-movement (non-REM) sleep
There are two stages of sleep; one is rapid-eye-movement (REM) sleep during which people dream, and the other is non-REM sleep during which the brain is at rest. Non-REM sleep and REM sleep occur alternately. It is known that PGD₂, which is synthesized by lipocalin-type PGDS abundant in encephalic liquid, induces natural non-REM sleep.

*4 Duchenne muscular dystrophy
Muscular dystrophy is an inherited progressive muscular atrophy. Duchenne muscular dystrophy is the most common but the symptom is the severest among all types of muscular dystrophy.

*5 Nuclear magnetic resonance (NMR) analysis
NMR analysis is one of the methods of examining molecules and atoms. Approximately 20% of the steric structures of proteins are determined by NMR analysis. Information on dynamic structure or relatively weak binding of molecules of proteins in the liquid state is obtained.

For more information, please contact:
Dr. Masashi Miyano, RIKEN SPring-8 Center
e-mail: mail ,

or
Dr. Yoshihiro Urade, Osaka Bioscience Institute
e-mail: mail .

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Flexible Structure of Prostaglandin D Synthase (PGDS) with Various Functions Supporting Sleep Induction, and Maturation and Maintenance of Reproductive System (Press Release)