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 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 1.90 Å resolution, reveals a two-domain organization of the module (domains A and B). A deep hydrophobic pocket with a single bound molecule of oleic acid 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.

Keywords: Interphotoreceptor matrix; Interphotoreceptor retinoid-binding protein; Oleic acid; RBP3; Retina; Visual cycle; X-ray structure; Zebrafish.

Figure 1. Alignment of the amino acid sequences of modules of IRBPs: Xenopus module 2 (xIRBP mod2), zebrafish (zIRBP) module 1 (mod1) and module 2 (mod2)

Functionally important substitutions are highlighted and color-coded.

Figure 2. A ribbon diagram of the crystal structure of zebrafish IRBP module 1

The N terminus is shown in blue and the C terminus in red. The domains and the secondary structure elements are marked. The oleic acid (OLA) binding site is shown with the bound oleic acid molecule. The terminal amino acids are also marked.

Figure 3. Superposition of the crystal structures of zebrafish IRBP module 1 (salmon) and Xenopus IRBP module 2 (blue)

The N-terminal domain A’s appear to be rotated roughly 21±4° from each other (resembling the rotation of the imaginary A-domain axis as shown) about the viewing axis of the figure at the domain interface.

Figure 4. Electron density (2F_obs−F_cal) omit map for the bound oleic acid (OLA) molecule in domain A

The electron density map is contoured at 1σ. Atoms C9 and C10 are labeled to indicate the location of the C9=C10 cis double bond.

Figure 5. Oleic acid binding site in domain A of zIRBP module 1

Important amino acid side chains inside the cavities and at the perimeter are shown. van der Waals surfaces of ligand and protein atoms are depicted in semi-transparent colors. The location of the C9=C10 cis double bond is marked by the labels 9 and 10.

Figure 6. Hydrophobic cavity in domain B

The semi-transparent van der Waals surface of the protein atoms is shown along with the protein backbone. Side chains that line the cavity are also shown.

Figure 7. Fluorescent spectroscopic titration of all-trans retinol binding to zIRBP (1μM)

Monitoring enhancement of all-trans retinol fluorescence in the presence of 0.0μM (■), 0.0022μM (●), 0.0112μM ▲), 0.02225μM (▼), 0.112μM (◆) of oleic acid.

Figure 8. Homology modeling of module 2 (z2, in cyan) after z1 (in salmon color)

Several small-to-large side chain substitutions at the cavity perimeter as well as within the interior in domain A are depicted. The location of oleic acid C9=C10 cis double bond is marked by the labels 9 and 10. But for some structural rearrangements, these substitutions are therefore likely to eliminate this oleic acid binding site from z2.