Crystal structure of native RPE65, the retinoid isomerase of the visual cycle

Abstract

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-Å 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.isomerization ͉ metalloprotein ͉ monotopic membrane protein V ision begins when the 11-cis-retinylidene chromophore of rhodopsin is photoisomerized to all-trans-retinylidene, a process resulting in receptor activation and transduction of the light signal (1). After rhodopsin is photoactivated, it is no longer responsive to light, so for vision to continue, a trans-to-cis isomerization mechanism must be present to regenerate lightsensitive visual pigments. In vertebrates, after photoisomerization, all-trans-retinylidene is hydrolyzed from rhodopsin, reduced to all-trans-retinol, and transported to a tissue adjacent to the photoreceptor layer known as the retinal pigment epithelium (RPE), where enzymatic isomerization occurs (2). An RPEspecific, microsomal membrane protein with an apparent molecular mass of 65 kDa, known as RPE65, was determined to be responsible for trans-to-cis retinoid isomerase activity in the RPE (3-5). The importance of this protein in visual function is also evident from the observation that certain RPE65 mutations cause a form of the hereditary childhood blinding disease known as Leber congenital amaurosis (LCA) or the less severe, lateronset disease, retinitis pigmentosa (RP) (6-8).Based on sequence homology, RPE65 belongs to a family of carotenoid cleavage oxygenase (CCO) enzymes that oxidatively cleave ␤-carotene or apocarotenoids (9-11). However, RPE65 is distinct from all other members of this family in that it simultaneously cleaves and isomerizes all-trans-retinyl esters to 11-cisretinol and a fatty acid rather than oxidatively cleaving carotenoids (3-5, 11, 12). Unlike the reactions catalyzed by other CCO family members, there is no obvious role for molecular oxygen in RPE65 enzymology. The only family member with a known crystal structure is an apocarotenoid oxygenase from Synechocystis that is 25% identical and 42% homologous to human RPE65 (13). All members of this fa...

Structure of RPE65 isomerase in a lipidic matrix reveals roles for phospholipids and iron in catalysis

“…Initial trials were conducted with concentrated extracts produced by treatment of 40 mg of RPE microsomes with 1 mL of buffered solution containing 20-30 mM C 8 E 6 . Surprisingly, we obtained a unique crystal form under conditions very similar to those previously reported for growth of hexagonal RPE65 crystals (13). These thin, plate-like crystals were extremely fragile and diffracted X-rays in a highly anisotropic manner, thus precluding their use for structure determination (Fig.…”

“…The general location of residues 110-126, which were completely disordered and thus not modeled in the previously reported RPE65 structure (13), is apparent in the lipid-embedded structure as a stretch of weak but mostly continuous electron density that runs parallel to the membrane surface (Fig. 3C).…”

“…5). Although the dimer interface is fairly hydrophobic and predicted to be stable in solution, it also features a number of intermolecular hydrogen bonds and salt bridges that could be labile in a high ionic strength environment (13). Support for the stability of the dimeric assembly was provided by additional crystal structures of RPE65 in space group P6 5 22 (Table 1, Crystal form B).…”

“…Historically, the inhibition of RPE retinoid isomerase activity by detergents was the major factor that delayed its link to RPE65, and the mechanism of inhibition remains incompletely understood. In the structure, we observed that residues composing the RPE65 membrane-interaction surface exhibit substantial disorder compared with the rest of the enzyme (11,13). Owing to the roles of these residues in membrane binding as well as substrate recognition and proper positioning of the substrate in the active site, detergent-induced alterations in their native structure, either by stripping the protein of its native phospholipids or by a direct effect, could inactivate the enzyme.…”

“…Previously, we determined the crystal structure of detergentsolubilized RPE65 purified from bovine RPE (13), which revealed a 4-His-ligated iron center contained within seven-bladed β-propeller architecture and a monotopic mode of membrane attachment. The structure featured residual active-site electron density that we speculated might represent a bound fatty acid molecule perhaps derived from a cleaved retinyl ester.…”