Structural Analysis of Lipocalin-Type Prostaglandin D Synthase Complexed with Prostaglandin J2

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS), a member of the lipocalin superfamily, is present in the brain. L-PGDS catalyzes the isomerization of PGH2 to produce PGD2, a potent endogenous somnogen. L-PGDS-produced PGD2 is sleep-inducing and accumulates in the brain during prolonged periods of wakefulness. We recently found that PGJ2, a metabolite of PGD2, inhibits the enzyme activity of L-PGDS and displays a much stronger inhibitory activity than other reported inhibitors, such as retinoids and AT-56. Therefore, information regarding the binding of the L-PGDS/PGJ2 complex should provide useful information for the development of drugs for treating sleep disorders. In this study, we report on an investigation of the three-dimensional structure of recombinant mouse L-PGDS complexed with PGJ2 by NMR and a proposed binding mechanism from the deduced molecular structure.Chemical shift assignments of the backbone and side-chain protons of L-PGDS/PGJ2 complex were done using two-dimensional 1H-15N HSQC, three-dimensional HNCACB, CBCA(CO)NH, HBHA(CO)NH, CCH-TOCSY, and HCCH-TOCSY. NOEs were collected from three-dimensional 13C-edited and 15N-edited NOESY experiments. In addition, to investigate the binding site of PGJ2, we performed NMR titration experiments and compared the HSQC of L-PGDS/PGJ2 complex with that of apo-L-PGDS. Significant changes in the chemical shifts of the catalytic Cys65 residue and other residues located in the bottom area of the binding pocket of L-PGDS were observed upon PGJ2 binding, indicating that PGJ2 competitively binds to the substrate binding site. NMR titration experiments further revealed that the exchange rate between the PGJ2 bound and unbound state is slow on the NMR time scale, suggesting that PGJ2 binds tightly to the L-PGDS with a dissociation constant in the nanomolar (nM) range.