Structure of zebrafish IRBP reveals fatty acid binding

Abstract

Interphotoreceptor retinoid-binding protein (IRBP) has a remarkable role in targeting and protecting all-trans and 11-cis retinol, and 11-cis retinal during the rod and cone visual cycles. Little is known about how the correct retinoid is efficiently delivered and removed from the correct cell at the required time. It has been proposed that different fatty composition at that the outer-segments and retinal-pigmented epithelium could have an important role is regulating the delivery and uptake of the visual cycle retinoids at the cell-interphotoreceptor-matrix interface. Although this suggests intriguing mechanisms for the role of local fatty acids in visual-cycle retinoid trafficking, nothing is known about the structural basis of IRBP-fatty acid interactions. Such regulation may be mediated through IRBP’s unusual repeating homologous modules, each containing about 300 amino acids. We have been investigating structure-function relationships of Zebrafish IRBP (zIRBP), which has only two tandem modules (z1 and z2), as a model for the more complex four-module mammalian IRBP’s. Here we report the first X-ray crystal structure of a teleost IRBP, and the only structure with a bound ligand. The X-ray structure of z1, determined at 1.90Å resolution, reveals a two-domain organization of the module (domains A and B). A deep hydrophobic pocket was identified within the N-terminal domain A. In fluorescence titrations assays, oleic acid displaced all-trans retinol from zIRBP. Our study, which provides the first structure of an IRBP with bound ligand, supports a potential role for fatty acids in regulating retinoid binding.

Fold conservation and proteolysis in zebrafish IRBP structure: Clues to possible enzymatic function?

“…The expression and purification of the full length zIRBP is described in a recent publication from our laboratories (Ghosh et al, 2015). The cDNA for IRBP gene II , the product of which is two tandem homologous modules of about 300 amino acids, was obtained by screening a cDNA library (Rajendran et al, 1996).…”

“…The full-length zIRBP cDNA was subcloned with the addition of the TEV protease recognition site immediately downstream (3′) to factor Xa recognition site and immediately upstream (5′) of the zIRBP gene in the pET-30 Xa/LIC vector. Expression of the protein in Escherichia coli BL21(DE3) and selenomethionine (Se-Met)-substituted zIRBP has been described (Ghosh et al, 2015). …”

“…The recombinant zIRBP was crystallized by the sitting-drop vapor diffusion at 23°C (Ghosh et al, 2015). Diffraction data on the crystals of zIRBP were collected at the synchrotron X-ray beamline 19ID, the Structural Biology Center, Advanced Photon Source, Argonne National Laboratory, Argonne, IL.…”

“…Further clues to the function of IRBP may come from its structure. Although IRBP does contain hydrophobic domains capable of binding retinoids (Gonzalez-Fernandez et al, 2007; Gonzalez-Fernandez et al, 2009) and fatty acids (Ghosh et al, 2015), what other structural motifs are present that could contribute to the functions summarized above?…”

“…During a recent X-ray crystallographic study of oleic acid-binding to zebrafish IRBP (zIRBP) a possible autoproteolytic activity was observed (Ghosh et al, 2015). In that study, during formation of holo-zIRBP crystals the protein was cleaved near its center.…”