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Federico Gonzalez-Fernandez, Thomas Bevilacqua, Kee-Il Lee, Reena Chandrashekar, Larson Hsu, Mary Alice Garlipp, Jennifer B. Griswold, Rosalie K. Crouch, Debashis Ghosh; Retinol-Binding Site in Interphotoreceptor Retinoid-Binding Protein (IRBP): A Novel Hydrophobic Cavity. Invest. Ophthalmol. Vis. Sci. 2009;50(12):5577-5586. doi: https://doi.org/10.1167/iovs.08-1857.
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Interphotoreceptor retinoid-binding protein (IRBP) appears to target and protect retinoids during the visual cycle. X-ray crystallographic studies had noted a ββα-spiral fold shared with crotonases and C-terminal protein transferases. The shallow cleft formed by the fold was assumed to represent the retinol-binding site. However, a second hydrophobic site consisting of a highly restricted cavity was more recently appreciated during in silico ligand-docking studies. In this study, the ligand-binding environment within the second module of Xenopus IRBP (X2IRBP) is defined.
Pristine recombinant polypeptide corresponding to X2IRBP was expressed in a soluble form and purified to homogeneity without its fusion tag. Phenylalanine was substituted for tryptophan at each of the putative retinol-binding domains (W450F, hydrophobic cavity; W587F, shallow cleft). Binding of 11-cis and all-trans retinol were observed in titrations monitoring retinol fluorescence enhancement, quenching of tryptophan fluorescence, and energy transfer. The effect of oleic acid on retinol binding was also examined.
A ligand-binding stoichiometry of ∼1:1 was observed for 11-cis and all-trans with K d in the tens of nanomolar range. The substitution mutants showed little effect on retinol binding in titrations after fluorescence enhancement. However, the W450F and not the W587F mutant showed a markedly reduced capacity for fluorescence quenching for both 11-cis and all-trans retinol. Oleic acid inhibited the binding of 11-cis and all-trans retinol in an apparent noncompetitive manner.
The binding site for 11-cis and all-trans retinol is a novel hydrophobic cavity that is highly restrictive and probably distinct from the long chain fatty acid–binding site.
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