Systematic interaction analysis of human lipocalin-type prostaglandin D synthase with small lipophilic ligands

Abstract

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.

Interaction of Multimicrobial Synthetic Inhibitor 1,2-Bis(2-Benzimidazolyl)-1,2-Ethanediol with Serum Albumin: Spectroscopic and Computational Studies

“…The fluorescence quenching titration method has been extensively used to elucidate the ligand binding behavior of proteins [16], [18], [28]. Figure 2A shows the 1,2-bis(2-benzimidazolyl)-1,2-ethanediol (BBE) induced quenching of bovine serum albumin (BSA) fluorescence at different temperatures.…”

Fine‐tuned broad binding capability of human lipocalin‐type prostaglandin D synthase for various small lipophilic ligands

“…The selection of proper binding systems is a critical point in characterizing intermolecular interactions using ITC. We recently performed in vitro binding studies of human lipocalin‐type prostaglandin (PG) D synthase (L‐PGDS, prostaglandin‐H2 D‐isomerase, EC 5.3.99.2) using the tryptophan fluorescence quenching, TNS competition assay and ITC [15]. Spectroscopic studies revealed that Human L‐PGDS could bind to a large range of lipophilic binding partners, including heme metabolites, retinoids, thyroids, steroids, flavonoids, and saturated fatty acids, which differ in molecular size and physico‐chemical properties [15].…”

“…We recently performed in vitro binding studies of human lipocalin‐type prostaglandin (PG) D synthase (L‐PGDS, prostaglandin‐H2 D‐isomerase, EC 5.3.99.2) using the tryptophan fluorescence quenching, TNS competition assay and ITC [15]. Spectroscopic studies revealed that Human L‐PGDS could bind to a large range of lipophilic binding partners, including heme metabolites, retinoids, thyroids, steroids, flavonoids, and saturated fatty acids, which differ in molecular size and physico‐chemical properties [15]. In addition, ITC results showed that L‐PGDS bound to two molecules of heme metabolites such as hemin, biliverdin, and bilirubin with high and low affinities [15].…”

“…To elucidate the relative contributions of driving forces to intermolecular interactions, we investigated the ITC‐based thermodynamics of 18 binding systems of L‐PGDS with 15 variations of ligands (retinoic acids, l ‐thyroxine, progesterone, genistein, and PGH 2 ‐analog U‐46619) including the 3 heme metabolites (hemin, biliverdin, and bilirubin) previously studied [15]. The results showed that the formation of all complexes was driven by enthalpy mainly due to intermolecular hydrogen bonding, and not always by entropic gains such as the typical concept of the hydrophobic effect.…”

Structural and dynamic insights into substrate binding and catalysis of human lipocalin prostaglandin D synthase

“…Interestingly, chicken and fi sh L-PGDS lack enzymatic activity and operate primarily as lipophilic ligand vehicles ( 20 ). Human L-PGDS binds to a range of all-trans -retinoic acids (RAs), biliverdin, bilirubin, and thyroid hormones with good affi nity ( 21 ). Hence, it has been proposed to be a vital RA transporter and scavenger for bile pigments ( 22,23 ).…”