Stabilized G protein binding site in the structure of constitutively active metarhodopsin-II
Edited by Brian K. Kobilka, Stanford University School of Medicine, Stanford, CA, and approved October 24, 2011 (received for review August 26, 2011)
Abstract
G protein-coupled receptors (GPCR) are seven transmembrane helix proteins that couple binding of extracellular ligands to conformational changes and activation of intracellular G proteins, GPCR kinases, and arrestins. Constitutively active mutants are ubiquitously found among GPCRs and increase the inherent basal activity of the receptor, which often correlates with a pathological outcome. Here, we have used the M257Y6.40 constitutively active mutant of the photoreceptor rhodopsin in combination with the specific binding of a C-terminal fragment from the G protein alpha subunit (GαCT) to trap a light activated state for crystallization. The structure of the M257Y/GαCT complex contains the agonist all-trans-retinal covalently bound to the native binding pocket and resembles the G protein binding metarhodopsin-II conformation obtained by the natural activation mechanism; i.e., illumination of the prebound chromophore 11-cis-retinal. The structure further suggests a molecular basis for the constitutive activity of 6.40 substitutions and the strong effect of the introduced tyrosine based on specific interactions with Y2235.58 in helix 5, Y3067.53 of the NPxxY motif and R1353.50 of the E(D)RY motif, highly conserved residues of the G protein binding site.
Data Availability
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 4A4M).
ACKNOWLEDGMENTS.
We thank Fritz Winkler for discussions on crystallographic refinement. We thank Reiner Vogel for the FTIR spectroscopic characterization under crystallization conditions. We thank the staff at the Macromolecular Crystallography group at the Swiss Light Source (SLS) for excellent support during data collection. We are grateful for financial support from the Swiss National Science Foundation (SNSF) Grant 31003A_132815 and the ETH Zürich within the framework of the National Center for Competence in Research in Structural Biology Program (to X.D, J.S. and G.F.X.S.). The work was further financially supported by National Institutes of Health Grant EY007965 (to D.D.O.).
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Cover image: Pictured are stems of the trembling aspen tree, Populus tremuloides, in San Juan National Forest, Colorado. William R. L. Anderegg et al. attempted to determine the potential mechanism behind the widespread drought-induced decline of trembling aspen forests across western North America. The authors report that the patterns of canopy and root death observed in this species are likely linked to hydraulic failure in the roots and branches. Hydraulic failure can impair water supply to the leaves and increase the trees’ water stress. See the article on pages 233–237. Image courtesy of Kimberly L. Pham (Stanford University).
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Data Availability
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 4A4M).
Submission history
Published online: December 23, 2011
Published in issue: January 3, 2012
Keywords
Acknowledgments
We thank Fritz Winkler for discussions on crystallographic refinement. We thank Reiner Vogel for the FTIR spectroscopic characterization under crystallization conditions. We thank the staff at the Macromolecular Crystallography group at the Swiss Light Source (SLS) for excellent support during data collection. We are grateful for financial support from the Swiss National Science Foundation (SNSF) Grant 31003A_132815 and the ETH Zürich within the framework of the National Center for Competence in Research in Structural Biology Program (to X.D, J.S. and G.F.X.S.). The work was further financially supported by National Institutes of Health Grant EY007965 (to D.D.O.).
Notes
This article is a PNAS Direct Submission.
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The authors declare no conflict of interest.
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