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Free backbone carbonyls mediate rhodopsin activation

Nature Structural & Molecular Biology volume 23, pages 738–743 (2016)Cite this article

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Abstract

Conserved prolines in the transmembrane helices of G-protein-coupled receptors (GPCRs) are often considered to function as hinges that divide the helix into two segments capable of independent motion. Depending on their potential to hydrogen-bond, the free C=O groups associated with these prolines can facilitate conformational flexibility, conformational switching or stabilization of the receptor structure. To address the role of conserved prolines in family A GPCRs through solid-state NMR spectroscopy, we focus on bovine rhodopsin, a GPCR in the visual receptor subfamily. The free backbone C=O groups on helices H5 and H7 stabilize the inactive rhodopsin structure through hydrogen-bonds to residues on adjacent helices. In response to light-induced isomerization of the retinal chromophore, hydrogen-bonding interactions involving these C=O groups are released, thus facilitating repacking of H5 and H7 onto the transmembrane core of the receptor. These results provide insights into the multiple structural and functional roles of prolines in membrane proteins.

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Figure 1: Side (left) and top-down (right) views of the crystal structure of the visual receptor rhodopsin (PDB 1U19 (ref.9)) showing the positions of Pro2155.50, Pro2676.50, Pro2917.38 and Pro3037.50.
Figure 2: REDOR NMR as a probe of hydrogen-bonding changes of carbonyl residues at the i − 4 positions of Pro2155.50, Pro2676.50, Pro2917.38 and Pro3037.50.
Figure 3: 13C-15N REDOR NMR experiments of Meta II in the presence or absence of the C-terminal Gα peptide of transducin.
Figure 4: Receptor activation leads to repacking of helices H5–H7 on the TM core of rhodopsin.

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Acknowledgements

This work was supported by the National Institutes of Health (NIH) grant GM41412 (to S.O.S.) We thank H. Sasaki and X. Zhou (Institute of Protein Research, Osaka University) for expression and purification of several of the 15N-13C-labeled rhodopsin samples, and J. Goncalves for preliminary experiments with the Gα peptide. We thank J. Nathans (Johns Hopkins University) for providing the HEK293S cell line.

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  1. Naoki Kimata and Andreyah Pope: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA

    Naoki Kimata, Andreyah Pope, Omar B Sanchez-Reyes, Markus Eilers, Martine Ziliox & Steven O Smith

  2. School of Biological Sciences, University of Essex, Essex, UK

    Chikwado A Opefi & Philip J Reeves

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Contributions

M.E., P.J.R. and S.O.S. conceived the study; N.K., A.P. and M.E. prepared samples; M.E. and M.Z. collected and analyzed NMR data; A.P. and O.B.S.-R. analyzed the protein database for proline interactions; C.A.O. constructed rhodopsin mutants; and N.K., A.P., P.J.R. and S.O.S. wrote the manuscript.

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Correspondence to Steven O Smith.

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Kimata, N., Pope, A., Sanchez-Reyes, O. et al. Free backbone carbonyls mediate rhodopsin activation. Nat Struct Mol Biol 23, 738–743 (2016). https://doi.org/10.1038/nsmb.3257

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