Cryo-EM structure of the rhodopsin-Gαi-βγ complex reveals binding of the rhodopsin C-terminal tail to the gβ subunit.

Ching-Ju Tsai,Filip Pamula,Henning Stahlberg,Xavier Deupi,Roger Jp Dawson,Ricardo Adaixo,Hugues Matile,Gebhard Schertler,Nicholas Mi Taylor,Jonas Muehle,Jacopo Marino,Inayatulla Mohammed,Shoji Maeda,Tilman Flock

doi:10.7554/elife.46041

Ching-Ju Tsai, Filip Pamula + Show 12 more

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https://doi.org/10.7554/elife.46041

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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.

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G protein-coupled receptors (GPCRs) are the most diverse class of integral membrane proteins with almost 800 members in humans
To obtain a rhodopsin-Gai1b1g1 heterotrimer (Gi) complex suitable for structural studies, we expressed in human embryonic kidney (HEK) cells the constitutively active mutant of bovine rhodopsin N2C/M257Y/D282C (Deupi et al, 2012) which binds to the Gi protein heterotrimer (Maeda et al, 2014)
The construct of the human Gai1 subunit was expressed in E. coli (Sun et al, 2015), while the Gb1g1 subunit was isolated from bovine retinae and contains native post-translational modification important for transducin function (Matsuda and Fukada, 2000)

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G protein-coupled receptors (GPCRs) are the most diverse class of integral membrane proteins with almost 800 members in humans. Nanobodies (Rasmussen et al, 2011), Fab fragments (Kang et al, 2018; Koehl et al, 2018), and mini-G proteins (Garcıa-Nafrıa et al, 2018b; Tsai et al, 2018) have been very important tools to overcome the inherent instability and flexibility of these complexes and obtain near atomic-resolution structures These structures represent snapshots of a particular state of the complex in the signaling cascade, and additional structural data are required to improve our understanding of this process (Capper and Wacker, 2018). This prompted us to perform a comparison of all available structures of GPCR-G protein complexes to generate a comprehensive contact map of this region We extended this analysis to the binding interface formed by the C-terminal helix a5 of Ga and found that only a few G protein subtype-specific residues consistently bind to the receptors. These contacts are ubiquitous anchoring points that may be involved in the selective engagement and activation of G proteins

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