Systematic interaction analysis of human lipocalin-type prostaglandin D synthase with small lipophilic ligands.
L-PGDS [lipocalin-type PG (prostaglandin) D synthase] is a multi-functional protein, acting as a PGD₂-producing enzyme and a lipid-transporter. In the present study, we focus on the function of L-PGDS as an extracellular transporter for small lipophilic molecules. We characterize the binding mechanism of human L-PGDS for the molecules, especially binding affinity stoichiometry and driving force, using tryptophan fluorescence quenching, ICD (induced circular dichroism) and ITC (isothermal titration calorimetry). The tryptophan fluorescence quenching measurements revealed that haem metabolites such as haemin, biliverdin and bilirubin bind to L-PGDS with significantly higher affinities than the other small lipophilic ligands examined, showing dissociation constant (K(d)) values from 17.0 to 20.9 nM. We focused particularly on the extra-specificities of haem metabolites and L-PGDS. The ITC and ICD data revealed that two molecules of the haem metabolites bind to L-PGDS with high and low affinities, showing K(d) values from 2.8 to 18.1 nM and from 0.209 to 1.63 μM respectively. The thermodynamic parameters for the interactions revealed that the contributions of enthalpy and entropy change were considerably different for each haem metabolite even when the Gibbs energy change was the same. Thus we believe that the binding energy of haem metabolites to L-PGDS is optimized by balancing enthalpy and entropy change.
Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan.
Satoshi
S
Kume
Young-Ho
YH
Lee
Yuya
Y
Miyamoto
Harumi
H
Fukada
Yuji
Y
Goto
Takashi
T
Inui
D019943:Amino Acid Substitution; D001663:Bilirubin / Q000737:chemistry / Q000378:metabolism*; D001664:Biliverdine / Q000737:chemistry / Q000378:metabolism*; D006427:Hemin / Q000737:chemistry / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D054834:Lipocalins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation; D058767:Protein Unfolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013816:Thermodynamics
D008024:Ligands; D054834:Lipocalins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D006427:Hemin; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase; D001664:Biliverdine; D001663:Bilirubin