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Crystal structure of metarhodopsin II

Abstract

G-protein-coupled receptors (GPCRs) are seven transmembrane helix (TM) proteins that transduce signals into living cells by binding extracellular ligands and coupling to intracellular heterotrimeric G proteins (Gαβγ)1. The photoreceptor rhodopsin couples to transducin and bears its ligand 11-cis-retinal covalently bound via a protonated Schiff base to the opsin apoprotein2. Absorption of a photon causes retinal cis/trans isomerization and generates the agonist all-trans-retinal in situ. After early photoproducts, the active G-protein-binding intermediate metarhodopsin II (Meta II) is formed, in which the retinal Schiff base is still intact but deprotonated. Dissociation of the proton from the Schiff base breaks a major constraint in the protein and enables further activating steps, including an outward tilt of TM6 and formation of a large cytoplasmic crevice for uptake of the interacting C terminus of the Gα subunit3,4,5. Owing to Schiff base hydrolysis, Meta II is short-lived and notoriously difficult to crystallize. We therefore soaked opsin crystals with all-trans-retinal to form Meta II, presuming that the crystal’s high concentration of opsin in an active conformation (Ops*)6,7 may facilitate all-trans-retinal uptake and Schiff base formation. Here we present the 3.0 Å and 2.85 Å crystal structures, respectively, of Meta II alone or in complex with an 11-amino-acid C-terminal fragment derived from Gα (GαCT2). GαCT2 binds in a large crevice at the cytoplasmic side, akin to the binding of a similar Gα-derived peptide to Ops* (ref. 7). In the Meta II structures, the electron density from the retinal ligand seamlessly continues into the Lys 296 side chain, reflecting proper formation of the Schiff base linkage. The retinal is in a relaxed conformation and almost undistorted compared with pure crystalline all-trans-retinal. By comparison with early photoproducts we propose how retinal translocation and rotation induce the gross conformational changes characteristic for Meta II. The structures can now serve as models for the large GPCR family.

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Figure 1: Structures of inactive rhodopsin, active Meta II and Meta II in complex with a Gα fragment.
Figure 2: Retinal binding pocket of Meta II.
Figure 3: Superposition of rhodopsin, Batho, Lumi and Meta II.
Figure 4: Conserved E(D)RY and NPxxY(x) 5,6 F regions.

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Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported structure have been deposited in the Protein Data Bank with the accession codes 3PQR and 3PXO.

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Acknowledgements

We thank J. Engelmann, C. Koch and B. Bauer for technical assistance, and F. Siebert and W. Hubbell for critically reading the manuscript. We are grateful to the European Synchrotron Radiation Facility (ESRF, Grenoble), D. von Stetten and A. Royant of the ID29S-Cryobench (ESRF, Grenoble) and U. Müller and the scientific staff of the BESSY-MX/Helmholtz Zentrum Berlin für Materialien und Energie at beamlines BL 14.1 and BL 14.2, where the data were collected, for continuous support. This work was supported by the DFG Sfb449 (to O.P.E.), Sfb740 (to O.P.E. and K.P.H.) and an Advanced Investigator ERC grant (to K.P.H.) and by the Canada Research Chairs Program (to E.F.P.). H.-W.C. gratefully acknowledges the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0002738) and CBNU funds for overseas research 2009. Y.J.K. thanks the Leibniz Graduate School of Molecular Biophysics, Berlin, for a scholarship.

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Contributions

H.-W.C., Y.J.K. and J.H.P. are joint first authors. H.-W.C., Y.J.K., J.H.P. performed preparation and crystallization of opsin/opsin−GαCT2. H.-W.C. performed the soaking experiment of both crystals. O.P.E. designed GαCT2. H.-W.C., Y.J.K., J.H.P., P.S., O.P.E. performed the data collection. Y.J.K., P.S., N.K. performed the structural analysis of Meta II, and J.H.P., P.S., E.F.P. performed the structural analysis of Meta II·GαCT2. T.M. performed the spectroscopic and biochemical analysis. H.-W.C., N.K., K.P.H., P.S., O.P.E. analysed data and H.-W.C., K.P.H., O.P.E. wrote the paper with contributions from all authors.

Corresponding authors

Correspondence to Hui-Woog Choe, Klaus Peter Hofmann or Oliver P. Ernst.

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The authors declare no competing financial interests.

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This file contains Supplementary Figures 1-10 with legends, Supplementary Tables 1-2, a Supplementary Discussion and additional references. (PDF 8968 kb)

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Choe, HW., Kim, Y., Park, J. et al. Crystal structure of metarhodopsin II. Nature 471, 651–655 (2011). https://doi.org/10.1038/nature09789

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