Crystal Structure of Native RPE65, the Retinoid Isomerase of the Visual Cycle

Sep. 1, 2010

Vertebrate vision is maintained by the retinoid (visual) cycle, a complex enzymatic pathway that operates in the retina to regenerate the visual chromophore, 11-cis-retinal. A key enzyme in this pathway is the microsomal membrane protein RPE65. This enzyme catalyzes the conversion of all-trans-retinyl esters to 11-cis-retinol in the retinal pigment epithelium (RPE). Mutations in RPE65 are known to be responsible for a subset of cases of the most common form of childhood blindness, Leber congenital amaurosis (LCA). Although retinoid isomerase activity has been attributed to RPE65, its catalytic mechanism remains a matter of debate. Also, the manner in which RPE65 binds to membranes and extracts retinoid substrates is unclear. To gain insight into these questions, we determined the crystal structure of native bovine RPE65 at 2.14-A resolution. The structural, biophysical, and biochemical data presented here provide the framework needed for an in-depth understanding of the mechanism of catalytic isomerization and membrane association, in addition to the role mutations that cause LCA have in disrupting protein function.

Figure RPE65 structure and topology. (A) Structure of an RPE65 monomer viewed from the bottom face of the seven-bladed β-propeller. Blades are numbered I through VII with the blade where "velcro" closure of the core propeller fold occurs labeled as blade VII in accord with established convention. Absolutely conserved His180, His241, His313, and His527 residues are shown as sticks coordinating the natively bound iron ion, shown as an orange sphere. (B) An RPE65 topology diagram showing the positions of residues involved in iron ion-coordination, as well as the position of the palmitoylated Cys residue. Numbers beside these residues indicate their position in the RPE65 amino acid sequence. (C) The proposed membrane-binding surface of RPE65. The mouth of the presumed substrate entry/product exit tunnel (shown as blue mesh) is surrounded by three groups of mainly hydrophobic residues, colored orange, that are likely responsible for anchoring RPE65 to RPE membranes. The maroon dashed line represents the approximate position of a disordered segment. A helical wheel plot of this segment (Inset) indicates that it may form an amphipathic α-helix under appropriate conditions.

Results from: Kiser, P., Golczak, M., Lodowski, D., Chance, M.R., Palczewski, K. Proc. Nat. Acad. Sci, 106(41):17325-30, 2009.