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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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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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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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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DOI: https://doi.org/10.1038/nsmb.3257
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