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.
-
Rhodopsin-Gi protein complexElectron Microscopy Data Bank, EMD-4598.
-
Antibody FAB fragment targeting Gi protein heterotrimerProtein Data Bank, 6QNK.
-
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.
Reviewing Editor
- Nikolaus Grigorieff, Janelia Research Campus, Howard Hughes Medical Institute, United States
Version history
- Received: February 12, 2019
- Accepted: June 26, 2019
- Accepted Manuscript published: June 28, 2019 (version 1)
- Version of Record published: July 15, 2019 (version 2)
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.
Metrics
-
- 4,992
- views
-
- 874
- downloads
-
- 53
- 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)
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
-
- Biochemistry and Chemical Biology
- Cell Biology
Mediator of ERBB2-driven Cell Motility 1 (MEMO1) is an evolutionary conserved protein implicated in many biological processes; however, its primary molecular function remains unknown. Importantly, MEMO1 is overexpressed in many types of cancer and was shown to modulate breast cancer metastasis through altered cell motility. To better understand the function of MEMO1 in cancer cells, we analyzed genetic interactions of MEMO1 using gene essentiality data from 1028 cancer cell lines and found multiple iron-related genes exhibiting genetic relationships with MEMO1. We experimentally confirmed several interactions between MEMO1 and iron-related proteins in living cells, most notably, transferrin receptor 2 (TFR2), mitoferrin-2 (SLC25A28), and the global iron response regulator IRP1 (ACO1). These interactions indicate that cells with high MEMO1 expression levels are hypersensitive to the disruptions in iron distribution. Our data also indicate that MEMO1 is involved in ferroptosis and is linked to iron supply to mitochondria. We have found that purified MEMO1 binds iron with high affinity under redox conditions mimicking intracellular environment and solved MEMO1 structures in complex with iron and copper. Our work reveals that the iron coordination mode in MEMO1 is very similar to that of iron-containing extradiol dioxygenases, which also display a similar structural fold. We conclude that MEMO1 is an iron-binding protein that modulates iron homeostasis in cancer cells.
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2’s requirement for activation.
