Cryo-EM structure of the adenosine A2A receptor coupled to an engineered heterotrimeric G protein

  1. Javier García-Nafría
  2. Yang Lee
  3. Xiaochen Bai
  4. Byron Carpenter
  5. Christopher G Tate  Is a corresponding author
  1. MRC Laboratory of Molecular Biology, United Kingdom

Abstract

The adenosine A2A receptor (A2AR) is a prototypical G protein-coupled receptor (GPCR) that couples to the heterotrimeric G protein GS. Here we determine the structure by electron cryo-microscopy (cryo-EM) of A2AR at pH 7.5 bound to the small molecule agonist NECA and coupled to an engineered heterotrimeric G protein, which contains mini-GS, the βγ subunits and nanobody Nb35. Most regions of the complex have a resolution of ~3.8 Å or better. Comparison with the 3.4 Å resolution crystal structure shows that the receptor and mini-GS are virtually identical and that the density of the side chains and ligand are of comparable quality. However, the cryo-EM density map also indicates regions that are flexible in comparison to the crystal structures, which unexpectedly includes regions in the ligand binding pocket. In addition, an interaction between intracellular loop 1 of the receptor and the β subunit of the G protein was observed.

Data availability

Structural data have been deposited in the PDB under the accession code 6gdg and in EMDB with accession code 4390

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Javier García-Nafría

    Structural Studies, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.
  2. Yang Lee

    Structural Studies, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.
  3. Xiaochen Bai

    Structural Studies, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.
  4. Byron Carpenter

    Structural Studies, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1712-3528
  5. Christopher G Tate

    Structural Studies, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    For correspondence
    cgt@mrc-lmb.cam.ac.uk
    Competing interests
    Christopher G Tate, is a consultant and shareholder of Heptares Therapeutics, and they also funded this work.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2008-9183

Funding

Medical Research Council (U105197215)

  • Christopher G Tate

European Research Council (EMPSI 339995)

  • Christopher G Tate

Heptares Therapeutics (n/a)

  • Christopher G Tate

Pfizer UK (n/a)

  • Christopher G Tate

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Werner Kühlbrandt, Max Planck Institute of Biophysics, Germany

Version history

  1. Received: February 14, 2018
  2. Accepted: May 2, 2018
  3. Accepted Manuscript published: May 4, 2018 (version 1)
  4. Version of Record published: May 21, 2018 (version 2)

Copyright

© 2018, García-Nafría et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 9,927
    views
  • 1,661
    downloads
  • 216
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Javier García-Nafría
  2. Yang Lee
  3. Xiaochen Bai
  4. Byron Carpenter
  5. Christopher G Tate
(2018)
Cryo-EM structure of the adenosine A2A receptor coupled to an engineered heterotrimeric G protein
eLife 7:e35946.
https://doi.org/10.7554/eLife.35946

Share this article

https://doi.org/10.7554/eLife.35946

Further reading

    1. Biochemistry and Chemical Biology
    2. Neuroscience
    Maximilian Nagel, Marco Niestroj ... Marc Spehr
    Research Article

    In most mammals, conspecific chemosensory communication relies on semiochemical release within complex bodily secretions and subsequent stimulus detection by the vomeronasal organ (VNO). Urine, a rich source of ethologically relevant chemosignals, conveys detailed information about sex, social hierarchy, health, and reproductive state, which becomes accessible to a conspecific via vomeronasal sampling. So far, however, numerous aspects of social chemosignaling along the vomeronasal pathway remain unclear. Moreover, since virtually all research on vomeronasal physiology is based on secretions derived from inbred laboratory mice, it remains uncertain whether such stimuli provide a true representation of potentially more relevant cues found in the wild. Here, we combine a robust low-noise VNO activity assay with comparative molecular profiling of sex- and strain-specific mouse urine samples from two inbred laboratory strains as well as from wild mice. With comprehensive molecular portraits of these secretions, VNO activity analysis now enables us to (i) assess whether and, if so, how much sex/strain-selective ‘raw’ chemical information in urine is accessible via vomeronasal sampling; (ii) identify which chemicals exhibit sufficient discriminatory power to signal an animal’s sex, strain, or both; (iii) determine the extent to which wild mouse secretions are unique; and (iv) analyze whether vomeronasal response profiles differ between strains. We report both sex- and, in particular, strain-selective VNO representations of chemical information. Within the urinary ‘secretome’, both volatile compounds and proteins exhibit sufficient discriminative power to provide sex- and strain-specific molecular fingerprints. While total protein amount is substantially enriched in male urine, females secrete a larger variety at overall comparatively low concentrations. Surprisingly, the molecular spectrum of wild mouse urine does not dramatically exceed that of inbred strains. Finally, vomeronasal response profiles differ between C57BL/6 and BALB/c animals, with particularly disparate representations of female semiochemicals.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Claudia D Consalvo, Adedeji M Aderounmu ... Brenda L Bass
    Research Article

    Invertebrates use the endoribonuclease Dicer to cleave viral dsRNA during antiviral defense, while vertebrates use RIG-I-like Receptors (RLRs), which bind viral dsRNA to trigger an interferon response. While some invertebrate Dicers act alone during antiviral defense, Caenorhabditis elegans Dicer acts in a complex with a dsRNA binding protein called RDE-4, and an RLR ortholog called DRH-1. We used biochemical and structural techniques to provide mechanistic insight into how these proteins function together. We found RDE-4 is important for ATP-independent and ATP-dependent cleavage reactions, while helicase domains of both DCR-1 and DRH-1 contribute to ATP-dependent cleavage. DRH-1 plays the dominant role in ATP hydrolysis, and like mammalian RLRs, has an N-terminal domain that functions in autoinhibition. A cryo-EM structure indicates DRH-1 interacts with DCR-1’s helicase domain, suggesting this interaction relieves autoinhibition. Our study unravels the mechanistic basis of the collaboration between two helicases from typically distinct innate immune defense pathways.