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F. Gonzalez-Fernandez, J. Armenia, M. Murray, J. Griswold, M. Garlipp, T. Loehfelm, D. Ghosh; Zebrafish Interphotoreceptor Retinoid-Binding Protein (zIRBP): X-Ray Crystal Structure & Function. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1302.
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© ARVO (1962-2015); The Authors (2016-present)
IRBP has a remarkable role in targeting, and protecting all-trans and 11-cis retinol, and 11-cis retinal during the visual cycle. Fatty acids regulate this transport in a complex manner. The mechanism for this regulation may be mediated through IRBP’s unusual 4-module structure. Each ~300 aa module contains two hydrophobic ligand-binding domains. To reduce this complexity, we examine zebrafish IRBP, which consists of only 2 modules.
ZIRBP was expressed in E coli using pET-30 Xa/LIC. A TEV-protease site facilitates removal of vector-amino acids. ZIRBP was over-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. ZIRBP including seleno-methionine derivatives with bound all-trans retinol and oleic acid were mixed with the reservoir solutions of 35-44% polyethylene glycol 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 Argonne National Lab.
LC-MS/MS confirmed the authenticity of ZIRBP. Ligand binding: Retinol fluorescence enhancement, N = 1.01 ± 0.06, Kd = 0.12 ± 0.04 µM; Protein quenching, N = 0.81 ± 0.22, Kd = 0.23 ± 0.14 µM. X-ray diffraction data sets at the selenium absorption edge peak and a remote points were collected at 1.90Å resolution and that at the inflection point was gathered at 2.30Å. The structure was determined and is being refined at 1.90Å. The present 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 the C-terminal domains are significantly shifted from one other. Electron densities at 2 possible retinol and/or fatty acid binding sites are being scrutinized. Interestingly, one of the sites is situated at the interface of the observed domain movement.
Significant structural changes suggest a mechanism for noncompetive interactions between hydrophobic ligand-binding domains.
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