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Federico Gonzalez-Fernandez, Molly Sprada, Debashis Ghosh; Structure of the Ligand-Binding Domain Suggests Novel Functions for Interphotoreceptor Retinoid-Binding Protein (IRBP) in the Zebrafish Retina. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2219.
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Retinoid binding by IRBP may be regulated by fatty acids (Wolf. G Nutr. Rev., Vol. 56, 1998). This regulation may be mediated through IRBP’s unusual 4 module structure. To reduce the complexity of the system, we examined zebrafish IRBP (zIRBP), which consists of only 2 modules. The IRBP single-module structural fold is evolutionally related to the crotonase/C-terminal peptidase (Cptase) family, which possesses enzymatic activity in diverse settings.
zIRBP was expressed in E coli using pET-30 Xa/LIC. zIRBP was expressed and purified by Ni2+-affinity and anion-exchange chromatography. All-trans retinol binding was characterized by fluorescence spectroscopy. zIRBP was crystallized by sitting-drop vapor diffusion. Solutions of zIRBP including seleno-methionine derivatives in the presence of molar excesses of all-trans retinol and oleic acid were mixed with the reservoir solutions of 35-44% PEG in 100mM Hepes containing 100mM NaBr in the 1:1, 2:1 and 3:1 volume ratios and vapor diffused against the reservoir solutions. The diffraction data was collected at the Advanced Photon Source, Argonne, IL.
Amino acid analysis confirmed the authenticity of zIRBP. Ligand binding: fluorescence enhancement, N = 1.01 ± 0.06, Kd = 0.12 ± 0.04 µM; quenching, N = 0.81 ± 0.22, Kd = 0.23 ± 0.14 µM. Data sets at the selenium absorption edge peak and a remote point were collected at 1.90Å and that at the inflection point were gathered at 2.30Å. The structure was determined and refined at 1.90Å. The R-factor for all reflections is 0.24 and the R-free value is 0.27. zIRBP was cleaved, possibly by autocatalysis, between the modules during the crystallization process, and only module 1 crystallized. The structure of holo-zIRBP was compared to the apo-Xenopus IRBP module 2 structure. Superposition shows that although the structures of these two IRBPs are homologous, their N- and C-terminal domains are shifted from one another. The e-density within the cavity is consistent with oleic acid having a mobile carboxylate end. Interestingly, the second site, housing residues similar to the structural homolog Cptase catalytic triad, is situated at the interface of the observed domain movement.
Our data supports mutatgenesis studies assigning the ligand-binding site to a hydrophobic cavity (Gonzalez-Fernandez et al. IVOS, Vol. 50. 2009). Under our experimental conditions, the site probably favors oleic acid binding. The altered inter-domain orientation suggests allostery between the two possible ligand sites. Potential protease-like activity at the second site is currently under study.
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