Docking model of PGH₂ to the catalytic pocket of L-PGDS (A) and the proposed catalytic mechanism of L-PGDS (B). See “Discussion” for detailed description.

Kinetics and binding of PGH₂ analogues to L-PGDS. A, reaction equation of PGDS from PGH₂ to PGD₂ and chemical structures of PGH₂ analogues U-44069 and U-46619. B, Lineweaver-Burk plots of the Δ^1–24-l-pgds activity inhibited by U-44069 and U-46619. Shown are sensograms (C) and the dose-response curves (D) of total (dashed lines), nonspecific (dotted lines), and specific (solid lines) binding of U-44069 (white circle) and U-46619 (black circle) to Δ^1–24-C65A L-PGDS, as assessed by the surface plasmon resonance analysis.

Comparison of the three-dimensional architectures of whole molecules of L-PGDS and H-PGDS (A) and of their catalytic pockets in stereo (B). A, l-pgds is shown on the left and H-PGDS on the right in rainbow-colored schematic diagrams with transparent molecular surfaces. Cys⁶⁵ in l-pgds and GSH bound to H-PGDS are shown as ball and stick models (black, carbon; red, oxygen; blue, nitrogen; yellow, sulfur). B, superimposed corresponding side chains involved in PGDS catalysis by l-pgds (stick model of pink-colored carbon atoms) and by H-PGDS (sky blue carbon atoms).

Superimposed models of the crystal structures of Δ^1–24-C65A L-PGDS with the NMR structures of the Δ^1–24-C89A/C186A L-PGDS (A) and with the SAXS molecular envelope of mouse L-PGDS (B). The crystal structures of the open (pink) and closed (sky blue) forms of Δ^1–24-C65A l-pgds in schematic models are superimposed on 15 NMR structures of the Δ^1–24-C89A/C186A l-pgds in hair ribbon models (PDB code 2E4J) (43) or a transparent surface model of mouse apo-l-pgds by SAXS (44). Phe³⁴, Trp⁵⁴, Pro¹¹⁰, and Cys⁸⁹–Cys¹⁸⁶ residues are represented by stick models with transparent spheres in A. (Color codes are the same as in Fig. 1A).

Activation of the thiol group of Cys⁶⁵ by the hydroxy cluster within the catalytic pocket of L-PGDS and comparison with the thiol activation of H-PGDS and the θ class of GSH transferases. A, thiol titration assay of Δ^1–24-l-pgds and its mutants at pH 4 with 2,2′-dithiodipyridine. The chromogenic reaction was monitored spectrophotometrically at 340 nm, as described under “Experimental Procedures.” B, hydrogen bond networks of l-pgds, H-PGDS/the σ class of GSH transferases (σGST), and the θ class of GSH transferases (θGST). C, amino acid residues responsible for thiol activation in l-pgds (pink carbon), H-PGDS (blue), and the θ class of GSH transferases (green) in stereo. (Other color codes are the same as in Fig. 1).

Crystal structures of L-PGDS with open and closed cavities. A, ribbon diagrams of the l-pgds structure. β-Strands and α-helices are labeled A-I and 1–3, respectively. The open and closed l-pgds structures of the P2₁2₁2₁ and C222₁ crystals were superimposed on an orthogonal view of the barrel. The conformational differences between open and closed forms are colored in sky blue and pink, respectively, in the E-F loop, Phe³⁴ of an N-terminal tail (N) and Gln⁸⁸ of the C-D loop, in which Phe³⁴ (F34), Trp⁵⁴ (W54), Gln⁸⁸ (Q88), Pro¹¹⁰ (P110), and His¹¹¹ (H111) are shown in a stick model form. The Phe³⁴ residue was defined in the closed form but disordered in the open form, unplugging the bottom of the β-barrel entry, whereas Gln⁸⁸ of the C-D loop was identified in the open form (Q88) but not in the closed form. C65A residue (C65A), a disulfide bond between Cys⁸⁹ and Cys¹⁸⁶ (C89/186), and two putative N-linked glycosylation sites, Asn⁵¹ (N51) and Asn⁷⁸ (N78) are also indicated as stick models (green, carbon; red, oxygen; blue, nitrogen). B, l-pgds structures in another view are shown after about 90° rotation of A from the vertical. C, another view of the open-closed l-pgds structures is represented after horizontal rotation of B. D, a view of the superimposed mobile E-F loops from the closed (pink) and open (sky blue) structures. Trp⁵⁴ and His¹¹¹ at the top of the barrel are shown as stick models. The Pro¹¹⁰ residue in the up and down conformations of pyrrolidine ring puckerings (upP110o in sky blue and downP110c in pink) is shown in the respective open and closed forms of the l-pgds structures. E and F, architectures of the mobile E-F loop in the open (E) and closed (F) forms. The non-bonding interactions are indicated by dashed lines. In F, Trp⁵⁴ in helix 2 of the Ω loop is also shown. G, open and closed lids of the l-pgds cavity are represented as a silver molecular surface. The open and closed E-F loops are colored in sky blue and pink, respectively (left and right panels, respectively). H, the upper hydrophilic compartment with several polar residues, including the C65A (C65A), Ser⁴⁵ (S45), Thr⁶⁷ (T67), Ser⁸¹ (S81), His¹¹⁶ (H116), Ser¹³³ (S133), Thr¹⁴⁷ (T147), and Tyr¹⁴⁹ (Y149) shown in stick-model form (green, carbon; red, oxygen; blue, nitrogen).