Retinol-Binding Site in Interphotoreceptor Retinoid-Binding Protein (IRBP): A Novel Hydrophobic Cavity

Abstract

Purpose

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.

Methods

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.

Results

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.

Conclusions

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.