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Catalytic mechanism of a retinoid isomerase essential for vertebrate vision

Abstract

Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive because of uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for the treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligand positioned in an adjacent pocket. With the geometry of the RPE65–substrate complex clarified, we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. These data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules.

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Figure 1: Role of RPE65 in the RPE-based visual cycle.
Figure 2: Structural analysis and inhibitory effects of retinoid-mimetic visual cycle modulators.
Figure 3: Crystal structure of RPE65 in complex with emixustat at 1.8-Å resolution.
Figure 4: Structural determinants of RPE65 ester cleavage and isomerization specificity.
Figure 5: Proposed mechanism of retinoid isomerization catalyzed by RPE65.

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Acknowledgements

We thank L.T. Webster Jr. for helpful comments on this manuscript. This work was supported by funding from US National Institutes of Health grants EY023948 (M.G.), EY009339 (K.P.), EY021126 (K.P.) and CA157735 (G.P.T.), Department of Veterans Affairs IK2BX002683 (P.D.K.) and National Science Foundation MCB-084480 (G.P.T.). Data for this study were measured at beamline X29 of the National Synchrotron Light Source. Financial support came principally from the Offices of Biological and Environmental Research and of Basic Energy Sciences of the US Department of Energy (DOE) and from the National Center for Research Resources (P41RR012408), the National Institute of General Medical Sciences (P41GM103473) of the National Institutes of Health and the National Institute of Biomedical Imaging and Bioengineering (P30-EB-09998). We thank the staff at the Advanced Photon Source Northeastern Collaborative Access Team beamlines, supported by a grant from the National Institute of General Medical Sciences (P41 GM103403), for assistance with collection of preliminary diffraction data. This research used resources of the Advanced Photon Source, a US DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. K.P. is John H. Hord Professor of Pharmacology.

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Authors

Contributions

P.D.K., J.Z., G.P.T. and K.P. conceived and designed experiments. P.D.K. purified and crystallized RPE65, solved and refined the structures and performed structural analyses. J.Z. performed in vitro and in vivo retinoid isomerization assays. M.B., Q.L. and G.P.T. carried out chemical synthesis. W.S. collected diffraction data. X.S. assisted with crystallization and crystal harvesting. M.G. assisted with in vitro and in vivo retinoid isomerization assays. P.D.K. wrote the paper. K.P. coordinated and oversaw the research project. All authors discussed the results and commented on the manuscript.

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Correspondence to Krzysztof Palczewski.

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Competing interests

K.P. and M.G. are inventors of US Patent No. 8722669, “Compounds and Methods of Treating Ocular Disorders,” and US Patent No. 20080275134, “Methods for Treatment of Retinal Degenerative Disease,” issued to Case Western Reserve University (CWRU), whose values may be affected by this publication. CWRU may license this technology for commercial development. K.P. was a member of the scientific board of Vision Medicine, Inc. involved in developing visual cycle modulators whose values may be affected by this publication.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–5. (PDF 4988 kb)

41589_2015_BFnchembio1799_MOESM439_ESM.mp4

Binding site locations and electron density maps for the retinoid-mimetic inhibitors, emixustat and MB-001, and palmitate. (MP4 11613 kb)

Catalytically important features of the RPE65 active site. (MP4 12720 kb)

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Kiser, P., Zhang, J., Badiee, M. et al. Catalytic mechanism of a retinoid isomerase essential for vertebrate vision. Nat Chem Biol 11, 409–415 (2015). https://doi.org/10.1038/nchembio.1799

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