Human Cellular Retinaldehyde-Binding Protein Has Secondary Thermal 9-Cis-Retinal Isomerase Activity

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/259313787 Human Cellular Retinaldehyde-Binding Protein Has Secondary Thermal 9-cis-Retinal Isomerase Activity Article in Journal of the American Chemical Society · January 2014 DOI: 10.1021/ja411366w · Source: PubMed CITATIONS READS 6 123 12 authors, including: Christin Bolze Rachel E. Helbling Vifor Pharma 13 PUBLICATIONS 21 CITATIONS 3 PUBLICATIONS 13 CITATIONS SEE PROFILE SEE PROFILE Arwen Pearson Guillaume Pompidor University of Hamburg European Molecular Biology Laboratory 78 PUBLICATIONS 1,083 CITATIONS 37 PUBLICATIONS 286 CITATIONS SEE PROFILE SEE PROFILE Some of the authors of this publication are also working on these related projects: Melanopsin View project AdPLA LRAT View project All content following this page was uploaded by Achim Stocker on 10 November 2017. The user has requested enhancement of the downloaded file. Article pubs.acs.org/JACS Human Cellular Retinaldehyde-Binding Protein Has Secondary Thermal 9-cis-Retinal Isomerase Activity † ‡ † § ∥ ⊥ ∇ Christin S. Bolze, , Rachel E. Helbling, Robin L. Owen, Arwen R. Pearson, Guillaume Pompidor, , ⊥ ⊥ ○ † # # Florian Dworkowski, Martin R. Fuchs, , Julien Furrer, Marcin Golczak, Krzysztof Palczewski, † † Michele Cascella,*, and Achim Stocker*, † ‡ Department of Chemistry and Biochemistry, and Graduate School for Cellular and Biomedical Sciences, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland § Diamond Light Source, Harwell Science and Innovation Campus, Didcot, OX11 0DE, United Kingdom ∥ Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, United Kingdom ⊥ Swiss Light Source, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland # Department of Pharmacology, School of Medicine, Case Western Reserve University, 2109 Adelbert Road, Cleveland, Ohio 44106-4965, United States *S Supporting Information ABSTRACT: Cellular retinaldehyde-binding protein (CRALBP) chaperones 11- cis-retinal to convert opsin receptor molecules into photosensitive retinoid pigments of the eye. We report a thermal secondary isomerase activity of CRALBP when bound to 9-cis-retinal. UV/vis and 1H NMR spectroscopy were used to characterize the product as 9,13-dicis-retinal. The X-ray structure of the CRALBP mutant R234W:9-cis-retinal complex at 1.9 Å resolution revealed a niche in the binding pocket for 9-cis-aldehyde different from that reported for 11-cis-retinal. Combined computational, kinetic, and structural data lead us to propose an isomerization mechanism catalyzed by a network of buried waters. Our findings highlight a specific role of water molecules in both CRALBP-assisted specificity toward 9-cis-retinal and its thermal isomerase activity yielding 9,13-dicis-retinal. Kinetic data from two point mutants of CRALBP support an essential role of Glu202 as the initial proton donor in this isomerization reaction. ■ INTRODUCTION group.12 Simultaneously, it preserves the light-sensitive cis- Retinal is the photon-absorbing partner of opsins in cone and chromophore from premature photoisomerization in the rod cells of vertebrate eye retina. Absorption of a photon is constantly illuminated eye. Because 11-cis-retinal is inherently − unstable, this chromophore is essentially unsuitable for coupled to cis trans photoisomerization in the retinylidene 13 moiety triggering conformational changes in the opsin core.1,2 sustained replacement therapy. A solution to this dilemma Regenerative trans-to-cis recycling occurs in the rod and cone is provided by the more conformationally stable 9-cis-retinal, cells and in the neighboring retinal pigment epithelium (RPE) which allows larger quantities of iso-rhodopsin to exist in and Müller cells, respectively.3,4 The key step in the canonical photoreceptors, thereby probably reducing the activity of the retinoid cycle.14 9-cis-Retinal has been successfully administered visual cycle is the trans-to-cis isomerization catalyzed by RPE65 15,16 in the RPE.5 Recently, DES1, another isomerase, has been to animal models to restore visual function. Despite the fact implicated in the alternative cone visual cycle.6 Both of these that the principal components of the classical visual cycle are enzymes can produce substantial quantities of cis-retinoids known, a satisfactory explanation for cis-retinoid redundancy in 6,7 retinoid-recycling has not been established at the molecular other than 11-cis-retinal in vitro. Their in vivo selectivity is 17 probably controlled by mass action through interacting level. A full structural understanding of both the CRALBP:11- retinoid-binding proteins.8,9 cis-retinal and the CRALBP:9-cis-retinal complexes and their Cellular retinaldehyde-binding protein (CRALBP) chaper- intermediates is therefore crucial for improving retinoid ones 11-cis-retinoids for the conversion of opsin receptor replacement therapies of inherited human blinding diseases. molecules into photosensitive visual pigments. CRALBP is a Numerous retinal pathologies are associated with mutations of water-soluble protein that specifically binds cis-retinoids with the human gene encoding CRALBP (RLBP1) including ffi ≈ autosomal recessive retinitis pigmentosa, night blindness, high a nity. Dissociation constants for 11-cis-retinal/ol (Kd ≈ 21 nM/53 nM) and 9-cis-retinal (Kd 51 nM) have been reported.10,11 By trapping these retinals, CRALBP protects the Received: June 2, 2013 retina from side reactions of the chemically reactive aldehyde Published: December 13, 2013 © 2013 American Chemical Society 137 dx.doi.org/10.1021/ja411366w | J. Am. Chem. Soc. 2014, 136, 137−146 Journal of the American Chemical Society Article Bothnia dystrophy, retinitis punctata, Newfoundland rod-cone once the medium reached an oxygen threshold of 12.5% and a pH of dystrophy, and the less severe phenotype of delayed “dark 6.83. After 24 h, cells were harvested by centrifugation at 5000g for 25 ” 18,19 min, and 50 g batches of cells were resuspended in 400 mL of lysis adaptation . ff We recently reported the crystal structures of both WT bu er (20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 20 mM imidazole). CRALBP and its Bothnia disease mutant R234W in complex Cells were disrupted by homogenization, and the lysate was 20 centrifuged at 50 000g for 30 min. (His)6-tagged proteins were with the highly abundant endogenous ligand 11-cis-retinal. fi ffi ’ puri ed from the supernatant by a nity chromatography on 20 mL of Here, we investigated the molecular basis of CRALBP s high Ni-NTA SUPERFLOW (Qiagen). The lysate was loaded onto the specificity for the less abundant natural isomer 9-cis-retinal and column, washed with 20 column volumes of lysis buffer, and purified report evidence for the first time that, when bound to protein was eluted in 70 mL of elution buffer (20 mM Tris-HCl, pH CRALBP, 9-cis-retinal undergoes trans−cis isomerization to 7.4, 100 mM NaCl, 200 mM imidazole). Protein concentrations in the 9,13-dicis-retinal. The process is thermally driven, and does not eluate were 3−4 mg/mL as determined by absorbance at 280 nm using require light absorption. To understand the mechanistic aspects NanoDrop (ThermoScientific). For crystallization experiments, the (His)6-tag was cleaved by adding 20 units of thrombin protease (GE of this phenomenon, we solved crystal structures of 9-cis-retinal ° in complex with both CRALBP and the Bothnia mutant Healthcare) to 20 mL of eluate at 4 C overnight, and the digested eluate was reloaded on Ni-NTA to remove the tag. Ligand loading of R234W. Analysis of these X-ray structures revealed the affinity purified CRALBP and the R234W mutant was carried out by presence of a binding mode for 9-cis-retinal distinct from that adding aliquots of a 20 mM 9-cis-retinal stock dissolved in ethanol to of the 11-cis conformer. In particular, binding of 9-cis-retinal the protein solutions at a 1.5 molar excess with a final ethanol was associated with the presence of a few ordered water concentration of 2% v/v. Mixtures were concentrated by a Vivaspin molecules in the binding cavity. These formed a hydrogen- 15R Hydrosart (Sartorius) to 30−50 mg/mL of protein with three bond network connecting the aldehyde group of retinal to the washes in low salt buffer (20 mM Tris-HCl, pH 7.4). Desalted samples side-chain of Glu202. Detection of a strong kinetic isotope were centrifuged at 16 000g for 10 min and loaded onto a self-packed 8 ff mL PorosHQ column (Applied Biosystems) previously equilibrated e ect in the process of isomerization leads us to propose a ff reaction mechanism involving catalytic proton shuttling. with low salt bu er. CRALBP proteins were eluted over a 40% v/v ’ gradient of high salt buffer (20 mM Tris-HCl, pH 7.4, 1 M NaCl). CRALBP s secondary activity as a thermal isomerase of 9-cis- Peak fractions representing monomeric ligand−protein complexes retinal yielding 9,13-dicis-retinal could imply a role in metabolic were concentrated and re-equilibrated in low salt buffer to a final pathways not directly associated with the visual cycle. concentration of 15 mg/mL of protein. Protein Crystallization and Data Collection. Hexagonal crystals ■ EXPERIMENTAL SECTION of wild-type CRALBP in complex with 9-cis-retinal were obtained at 18 Materials. 11-cis-Retinal was obtained from the National Eye °C by hanging-drop vapor-diffusion. Drops were set up by mixing Institute, National Institutes of Health (NEI/NIH), and 9-cis-retinal either 1, 2, or 3 μL of CRALBP:9-cis-retinal (15 mg/mL) with 1 μLof was purchased from Sigma-Aldrich. Retinoids were dissolved in reservoir solution (1.2 M K/Na tartrate, 0.2 M Li2SO4, 0.1 M Tris- − ° μ ethanol and kept at 20 C. D2O as well as CD3Cl were obtained HCl, pH 8.5) and were equilibrated against an 80 L reservoir from Cambridge Isotope Laboratories. Solvents for HPLC and solution. Crystals of 0.3 × 0.07 × 0.07 mm size were obtained after 3 spectroscopic experiments were of HPLC grade or better.

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