Design of tailor-made micro-carrier protein having molecular-selective recognition.

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Small-angle X-ray scattering (SAXS) of lipocalin-type prostaglandin D synthase (L-PGDS) and two other lipocalins, β-lactoglobulin (βLG) and retinal-binding protein (RBP) were measured to clarify their conformational changes induced by binding of small lipophilic ligands, such as all-trans-retinoic acid (RA), bilirubin (BR) and biliverdin (BV). The radius of gyration (R_g) was estimated to be 19.4Å for L-PGDS, and 18.8Å for L-PGDS/RA, 17.3Å for L-PGDS/BR and 17.8Å for L-PGDSn3V complexes, indicating that L-PGDS became compact after binding of these ligands. Alternatively, the R_g of βLG and RBP (20.3 and 26.2Å, respectively) was almost unchanged before and after binding of RA. Molecular modeling of L-PGDS based on the SAXS data indicated that the ligand binding changed the peripheral region of the molecule and suggested that such structural flexibility of the L-PGDS molecule is responsible for the broader ligand selectivity of L-PGDS than other lipocalins.The three-dimensional structure of recombinant mouse L-PGDS was determined by NMR. The structure of L-PGDS exhibited the typical lipocalin-fold, consisting of an 8-stranded, antiparallel β-barrel and a α-helix associated with the outer surface of the barrel. The interior of the barrel formed a hydrophobic cavity opening to the upper end of the barrel, the size of which was larger than those of other lipocalins and the cavity contained two pockets. Molecular docking studies based on the result of NMR experiments with RA and PGH_2 analog revealed that PGH_2 fully occupied the hydrophilic pocket 1, where Cys65 was located, and RA did the hydrophobic pocket 2, where amino acid residues important for retinoid-binding in other lipocalins were well conserved. These results indicated that the two binding sites in the large cavity of L-PGDS was responsible for the broad ligand specificity of L-PGDS and the non-competitive inhibition of L-PGDS activity by RA.