March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Structure of the Ligand-Binding Domain Suggests Novel Functions for Interphotoreceptor Retinoid-Binding Protein (IRBP) in the Zebrafish Retina
Author Affiliations & Notes
  • Federico Gonzalez-Fernandez
    Research Service, VAWNYHS, Amherst, New York
    Ophthalmology and Pathology & Anatomic Sciences, University at Buffalo/SUNY and SUNY Eye Institute, Buffalo, New York
  • Molly Sprada
    Research Service, VAWNYHS, Amherst, New York
    Ophthalmology and Pathology & Anatomic Sciences, University at Buffalo/SUNY and SUNY Eye Institute, Buffalo, New York
  • Debashis Ghosh
    Pharmacology, SUNY Upstate Medical University, Syracuse, New York
  • Footnotes
    Commercial Relationships  Federico Gonzalez-Fernandez, None; Molly Sprada, None; Debashis Ghosh, None
  • Footnotes
    Support  NIH/NEI grant EY09412 (D.G./F.G-F.), Veterans Affairs R & D Merit Review Award I01BX007080 (F.G.-F.), and an Unrestricted Grant to the SUNY Ross Eye Institute from Research to Prevent Blindness, Inc., N
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 2219. doi:
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    • Get Citation

      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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: extracellular matrix • photoreceptors • retinoids/retinoid binding proteins 
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