Cryo-EM structure of the rhodopsin-Gαi-βγ complex reveals binding of the rhodopsin C-terminal tail to the Gβ subunit
- Paul Scherrer Institute, Switzerland
- University of Basel, Switzerland
- Hoffmann-La Roche Ltd, Switzerland
Abstract
One of the largest membrane protein families in eukaryotes are G protein-coupled receptors (GPCRs). GPCRs modulate cell physiology by activating diverse intracellular transducers, prominently heterotrimeric G proteins. The recent surge in structural data has expanded our understanding of GPCR-mediated signal transduction. However, many aspects, including the existence of transient interactions, remain elusive. We present the cryo-EM structure of the light-sensitive GPCR rhodopsin in complex with heterotrimeric Gi. Our density map reveals the receptor C-terminal tail bound to the Gβ subunit of the G protein, providing a structural foundation for the role of the C-terminal tail in GPCR signaling, and of Gβ as scaffold for recruiting Gα subunits and G protein-receptor kinases. By comparing available complexes, we found a small set of common anchoring points that are G protein-subtype specific. Taken together, our structure and analysis provide new structural basis for the molecular events of the GPCR signaling pathway.
Data availability
The cryo-EM density map of the rhodopsin-Gi complex bound to Fab16 has been deposited in the EM Data Bank (accession code EMD-4598), and the related structure coordinates have been deposited in the Protein Data Bank (accession code 6QNO). The crystal structure of Fab16 has been deposited in the Protein Data Bank (accession code 6QNK). Source data for Figure 3 is provided in Suppl. Table 3.
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Rhodopsin-Gi protein complexElectron Microscopy Data Bank, EMD-4598.
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Antibody FAB fragment targeting Gi protein heterotrimerProtein Data Bank, 6QNK.
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Crystal structure of the rhodopsin-mini-Go complexProtein Data Bank, 6FUF.
Article and author information
Author details
Nicholas MI Taylor
Funding
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (310030_153145)
- Gebhard Schertler
Swiss Nanoscience Institute (A13.12 NanoGhip)
- Gebhard Schertler
National Centres of Competence in Research (TransCure)
- Henning Stahlberg
Holcim Stiftung
- Jacopo Marino
ETH Zurich
- Tilman Flock
University of Cambridge
- Tilman Flock
Roche
- Shoji Maeda
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (160805)
- Xavier Deupi
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (310030B_173335)
- Gebhard Schertler
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2019, Tsai 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.
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Cryo-EM structure of the rhodopsin-Gαi-βγ complex reveals binding of the rhodopsin C-terminal tail to the Gβ subunit
eLife 8:e46041.
https://doi.org/10.7554/eLife.46041
Further reading
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- Biochemistry and Chemical Biology
N 6,2’-O-dimethyladenosine (m6Am) is a modified nucleotide located at the first transcribed position in mRNA and snRNA that is essential for diverse physiological processes. m6Am mapping methods assume each gene uses a single start nucleotide. However, gene transcription usually involves multiple start sites, generating numerous 5’ isoforms. Thus, gene-level annotations cannot capture the diversity of m6Am modification in the transcriptome. Here, we describe CROWN-seq, which simultaneously identifies transcription-start nucleotides and quantifies m6Am stoichiometry for each 5’ isoform that initiates with adenosine. Using CROWN-seq, we map the m6Am landscape in nine human cell lines. Our findings reveal that m6Am is nearly always a high stoichiometry modification, with only a small subset of cellular mRNAs showing lower m6Am stoichiometry. We find that m6Am is associated with increased transcript expression and provide evidence that m6Am may be linked to transcription initiation associated with specific promoter sequences and initiation mechanisms. These data suggest a potential new function for m6Am in influencing transcription.
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- Biochemistry and Chemical Biology
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Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.
