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

Abstract

23G protein-coupled receptors (GPCRs) are the largest class of integral membrane proteins and 24represent key targets for pharmacological research. GPCRs modulate cell physiology by 25 engaging and activating a diversity of intracellular transducers, prominently heterotrimeric G 26 proteins, but also G protein-receptor kinases (GRKs) and arrestins. The recent surge in the 27 number of structures of GPCR-G protein complexes has expanded our understanding of G 28 protein recognition and GPCR-mediated signal transduction. However, many aspects of these 29 mechanisms, including the existence of transient interactions with transducers, have remained 30 elusive. 31Here, we present the cryo-EM structure of the light-sensitive GPCR rhodopsin in complex with 32 heterotrimeric Gi. In contrast to all reported structures, our density map reveals the receptor 33 C-terminal tail bound to the Gβ subunit of the G protein heterotrimer. This observation provides 34 a structural foundation for the role of the C-terminal tail in GPCR signaling, and of Gβ as 35 scaffold for recruiting Gα subunits and GRKs. By comparing all available complex structures, 36we found a small set of common anchoring points that are G protein-subtype specific. Taken 37 together, our structure and analysis provide new structural basis for the molecular events of 38 the GPCR signaling pathway.

Functional Optimization of Light-Activatable Opto-GPCRs: Illuminating the Importance of the Proximal C-terminus in G-protein Specificity

“…We started by exploring chimeras between bRhod (GenBank accession number: AH001149.2) and b2AR (GenBank accession number: AY136741.1), since (1) the proximal C-terminus of bRhod was hypothesized to be involved in G-protein selectivity (Tsai et al, 2019), (2) abundant structural data is available, (3) both belong to Class A GPCRs facilitating sequence alignment (Kleinlogel, 2016) and (4) since functional rhodopsin chimeras with all intracellular domains exchanged have previously been successfully engineered (Airan et al, 2009; Kim et al, 2005; Tichy et al, 2022; Yamashita et al, 2001). Using the available structures as templates, we successively replaced IL2, IL3 and the C-terminus of rhodopsin by homologous domains of b2AR (Fig.…”

“…The structural components and molecular processes of GPCR-G-protein interactions were first elucidated at the structural level in bovine rhodopsin (bRhod) (Salom et al, 2006) and the beta2-adrenoceptor (b2AR) (Rasmussen, DeVree, et al, 2011; Rosenbaum et al, 2009), whereas mGluR structures were only recently resolved (Seven et al, 2021) and the crystal structure of melanopsin remains to be elucidated. From the structural studies it became obvious that the intracellular loop 3 (IL3) (Ma et al, 2020; Rasmussen, DeVree, et al, 2011), the intracellular loop 2 (IL2) (Kang et al, 2018; Qiao et al, 2020; Seven et al, 2021; Tsai et al, 2019) as well as the C-terminus (Bertheleme et al, 2013; Seven et al, 2021; Tsai et al, 2019; Tsai et al, 2018) contact the G-protein. However, contact to the G-protein does not necessarily indicate that the domain mediates G-protein selectivity, since the multiple ionic interaction networks also comprising the GPCR core structure could mediate specific conformational differences required to accommodate a particular G-protein (Venkatakrishnan et al, 2016).…”

“…In group A GPCRs, the displacement of TM6 upon activation creates a cleft between TM3, TM5 and TM6 for insertion of the G-protein’s C-terminus. IL3 as a main contact point of the G-protein’s C-terminus is therefore considered to play a major role in G-protein selectivity (Rasmussen, DeVree, et al, 2011; Tsai et al, 2019; Zhou et al, 2019). This assumption was supported by functional studies with engineered chimeric Opto-GPCRs (Airan et al, 2009; Kim et al, 2005; Siuda et al, 2015; van Wyk et al, 2015).…”

“…H8 holds a crucial position towards the intracellular side, where G-protein coupling occurs, and was shown to actively promote G-protein binding as well as stabilizing conformational states of the GPCR (Bruno et al, 2012; Palczewski et al, 2000). Importantly, the short stretch between TM7 and H8 and the proximal C-terminus beyond H8 were structurally shown to be in close contact with the G-protein and predicted to determine selective G protein activation in bovine rhodopsin (Tsai et al, 2019; Tsai et al, 2018).…”

Dimeric Rhodopsin R135L Mutant-Transducin-like Complex Sheds Light on Retinitis Pigmentosa Misfunctions

“…The selection consisted in taking a dimeric S3 structure in which the monomers had ILD, RKD, and MIA values close to the average values obtained in the simulations with non-complex dimers (Tables 1 and 2). To set up the complexes with Gtl we considered the structure of the recently reported complex bovRHO-transducin heterotrimer Gtl-, which was solved by cryo-EM image reconstruction and solved at 4.38 Å (PDB code 6QNO) 8 . In this experimental structure the constitutively active mutant of bovRHO N2C/M257Y/D282C is in the monomeric form.…”