title: Structural and dynamic insights into substrate binding and catalysis of human lipocalin prostaglandin D synthase.
abstract: Lipocalin prostaglandin D synthase (L-PGDS) regulates synthesis of an important inflammatory and signaling mediator, prostaglandin D2 (PGD2). Here, we used structural, biophysical, and biochemical approaches to address the mechanistic aspects of substrate entry, catalysis, and product exit of this enzyme. Structure of human L-PGDS was solved in a complex with a substrate analog (SA) and in ligand-free form. Its catalytic Cys 65 thiol group was found in two different conformations, each making a distinct hydrogen bond network to neighboring residues. These help in elucidating the mechanism of the cysteine nucleophile activation. Electron density for ligand observed in the active site defined the substrate binding regions, but did not allow unambiguous fitting of the SA. To further understand ligand binding, we used NMR spectroscopy to map the binding sites and to show the dynamics of protein-substrate and protein-product interactions. A model for ligand binding at the catalytic site is proposed, showing a second binding site involved in ligand exit and entry. NMR chemical shift perturbations and NMR resonance line-width alterations (observed as changes of intensity in two-dimensional cross-peaks in [¹H,¹⁵N]-transfer relaxation optimization spectroscopy) for residues at the Ω loop (A-B loop), E-F loop, and G-H loop besides the catalytic sites indicate involvement of these residues in ligand entry/egress.
authors.affiliation: Division of Structural Biology and Biochemistry, Nanyang Technological University, Singapore; and; Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Sing Mei
authors.initials: SM
authors.lastname: Lim
authors.affiliation: Division of Structural Biology and Biochemistry, Nanyang Technological University, Singapore; and.
authors.forename: Dan
authors.initials: D
authors.lastname: Chen
authors.affiliation: Division of Structural Biology and Biochemistry, Nanyang Technological University, Singapore; and.
authors.forename: Hsiangling
authors.initials: H
authors.lastname: Teo
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Annette
authors.initials: A
authors.lastname: Roos
authors.affiliation: Division of Structural Biology and Biochemistry, Nanyang Technological University, Singapore; and.
authors.forename: Anna Elisabet
authors.initials: AE
authors.lastname: Jansson
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Tomas
authors.initials: T
authors.lastname: Nyman
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Lionel
authors.initials: L
authors.lastname: Trésaugues
authors.affiliation: Division of Structural Biology and Biochemistry, Nanyang Technological University, Singapore; and. Electronic address: KPERVUSHIN@ntu.edu.sg.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Pervushin
authors.affiliation: Division of Structural Biology and Biochemistry, Nanyang Technological University, Singapore; and; Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. Electronic address: PNORDLUND@ntu.edu.sg.
authors.forename: Pär
authors.initials: P
authors.lastname: Nordlund
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000737:chemistry*; D054834:Lipocalins / Q000737:chemistry*; D056004:Molecular Dynamics Simulation; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D054834:Lipocalins; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase
keywords: X-ray crystallography; lipid signaling; lipophilic substrate; nuclear magnetic resonance spectroscopy; protein-lipid interaction; β trace protein