G protein-coupled receptors (GPCRs) are the most diverse class of integral membrane proteins with almost 800 members in humans. GPCRs are activated by a great diversity of extracellular stimuli including photons, neurotransmitters, ions, proteins, and hormones (Glukhova et al., 2018). Upon activation, GPCRs couple to intracellular transducers, including four subtypes of G proteins (Gαs, Gαi/o, Gαq/11, Gα12/13) (Milligan and Kostenis, 2006), seven subtypes of GPCR kinases (GRKs) (Gurevich et al., 2012), and four subtypes of arrestins (Smith and Rajagopal, 2016) (Figure 1A), among many other partners (Magalhaes et al., 2012). While most GPCRs are promiscuous and can couple to more than one G protein subtype (Flock et al., 2017), the molecular determinants of G protein recognition are not yet fully understood. Understanding the molecular basis for G protein coupling and selectivity could lead to the design of drugs that promote specific signaling pathways and avoid unwanted side effects (Hauser et al., 2017).

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The recent surge in the number of structures of GPCR-G protein complexes has greatly expanded our understanding of G protein recognition and GPCR-mediated signal transduction. Out of the 13 structures of GPCR-G protein complexes available, six contain a Gi/o subtype: μ-opioid receptor bound to Gi (Koehl et al., 2018), adenosine A₁ receptor bound to Gi (Draper-Joyce et al., 2018), cannabinoid receptor one bound to Gi (Krishna Kumar et al., 2019), human rhodopsin bound to Gi (Kang et al., 2018), 5HT_1B receptor bound to Go (García-Nafría et al., 2018b), and bovine rhodopsin bound to an engineered Go (mini-Go) (Tsai et al., 2018). However, the preparation of GPCR-G protein complexes for structural biology still remains challenging (Munk et al., 2019). Nanobodies (Rasmussen et al., 2011), Fab fragments (Kang et al., 2018; Koehl et al., 2018), and mini-G proteins (García-Nafría 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. Importantly, these structures represent snapshots of a particular state of the complex in the signaling cascade, and therefore additional structural data are required to improve our understanding of this process (Capper and Wacker, 2018).

The photoreceptor rhodopsin is one of the best-characterized model systems for studying GPCRs, providing invaluable information on how receptor activation is translated into G protein and arrestin binding and activation (Hofmann et al., 2009; Hofmann and Palczewski, 2015; Scheerer and Sommer, 2017). Rhodopsin has been shown to interact with the Gβγ subunit of the G protein heterotrimer to assist binding and activation of the Gα subunit (Herrmann et al., 2004; Herrmann et al., 2006). After dissociation of the Gαβγ heterotrimer, the Gβ subunit recruits GRKs to the membrane, resulting in the phosphorylation of the receptor C-terminal tail (C-tail) (Claing et al., 2002; Pao and Benovic, 2002; Pitcher et al., 1998) and binding of arrestin (Goodman et al., 1996). Despite this biochemical evidence, a direct interaction between rhodopsin and Gβγ could not be observed in the existing complex (Kang et al., 2018).

Here, we present the cryo-EM structure of bovine rhodopsin in complex with a heterotrimeric Gi. Overall, our structure agrees well with current published structures (Kang et al., 2018; Tsai et al., 2018). Remarkably, the EM density map provides structural evidence for the interaction between the C-tail of the receptor and the Gβ subunit. The density map also shows that intracellular loops (ICL) 2 and 3 of rhodopsin are at contact distance to Gα. 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 then extended this analysis to the binding interface formed by the C-terminal helix α5 of Gα 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 also involved in the selective engagement and activation of G proteins.

To obtain a rhodopsin-Gi complex suitable for structural studies, we expressed in 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 Gαi1 subunit was expressed in E. coli (Sun et al., 2015), while the Gβ1γ1 subunit was isolated from bovine retinae and thus contains native post-translational modification important for transducin function (Matsuda and Fukada, 2000). We reconstituted the rhodopsin-Gαi1β1γ1 complex in the detergent lauryl maltose neopentyl glycol (LMNG) (Maeda et al., 2014) in presence of Fab16 (Maeda et al., 2018) (Figure 1—figure supplement 1), as cryo-EM screening revealed that the complex without Fab could not be refined to high resolution (Figure 1—figure supplements 2 and 3). During image processing (Figure 1—figure supplements 3 and 4), 3D classification revealed that the density corresponding to Gα is heterogeneous (Figure 1—figure supplement 5), particularly at flexible regions such as the α-helical (AH) domain. The AH domain was then excluded by using a soft mask during refinement, resulting in a map with a nominal global resolution of 4.38 Å (Figure 1—figure supplement 3D and E). The EM map was used to build a model of the complex (Figure 1, Figure 1—figure supplement 6).

Interaction between the C-terminal tail of rhodopsin and Gβ

The EM map reveals a density on the Gβ subunit as continuation of H8 of the receptor (Figure 2A), which corresponds to the C-tail of rhodopsin. This feature has only been observed in the recent structure of the M1 muscarinic acetylcholine receptor in complex with G11 (Maeda et al., 2019). We modeled half of the C-tail of rhodopsin (12 out of 25 residues; 324–335) into this density as a continuous stretched peptide with residues G324, P327 and G329 serving as flexible hinges (Figure 2B). This allows us to compare the structure of this region in three conformational states of the receptor: inactive (Okada et al., 2004), G protein-bound (this structure), and arrestin-bound (Zhou et al., 2017) (Figure 2B).

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In the inactive state, the C-tail folds around the cytoplasmic side of rhodopsin, although this is likely due to crystal packing as this region is intrinsically disordered in the absence of a binding partner (Jaakola et al., 2005; Venkatakrishnan et al., 2014).

In our G protein complex, the C-tail stretches over a polar surface on the cleft between Gα and Gβ, interacting with both subunits (Figure 2C and D). In Gβ, the interacting residues are C271, D290, and D291 in blade six and R314 in blade 7 of the β-propeller (Figure 2D), which impart a local negative electrostatic potential (Figure 2—figure supplement 1). These residues are conserved in Gβ1–4 (Figure 2—figure supplement 2B) but not in Gβ5, the least similar to the other Gβ subtypes (Dupré et al., 2009). In Gα, the interacting residues with the C-tail are N256H3.15 and K257H3.16 in helix 3 of the Ras domain (Figure 2D), which confer instead a local positive electrostatic potential (Figure 2—figure supplement 1). These residues are quite conserved across G protein subtypes except Gq (Figure 2—figure supplement 2A). Interestingly, these regions in Gα and Gβ that contact the receptor C-tail are also involved in recognition of GRK2 (Tesmer et al., 2005).

In the rhodopsin-arrestin complex part of the proximal segment of the C-terminus (residues 325–329) is not resolved, but the distal part could be modeled up to S343, eight residues longer than in our G protein complex and including two phosphorylated sites. In the presence of arrestin, the C-tail stretches further, with residues K339-T342 forming a short β-strand antiparallel to the N-terminal β-strand of arrestin (Figure 2B).

Thus, it appears that distinct portions of the C-tail are responsible for contacting different intracellular partners. The central part of the C-tail (residues 330–335) can bind to Gα and Gβ, while the distal half of the C-tail (residues 336–343), which contains five (out of six) phosphorylation sites (Azevedo et al., 2015), binds to arrestin (Figure 2C).

Comparison of the GPCR-G protein binding interface

As in the other existing GPCR-G protein complex structures, the C-terminal α5 helix of Gα forms the major contact interface to rhodopsin. The α5 helix consists of the last 26 amino acids of Gα (H5.01–26), in which the last five residues fold into a hook-like structure (Tsai et al., 2018). The majority of the contacts formed by the α5 helix to GPCRs concentrate in the region from H5.11 to H5.26 (Glukhova et al., 2018) (Figure 3—figure supplement 1).

We aligned the structures of available complexes using the Cα atoms of H5.11–26. This alignment reduces apparent differences in the binding positions and provides the ‘viewpoint of the G protein’ (Figure 3A). We compiled an exhaustive list of the residue-residue contacts between the receptor and the α5 helix in all the available GPCR/G protein complexes, and observed that the main contacts to the α5 helix are formed by TM5 and TM6, followed by TM3 and TM7/8 (Figure 3B, Figure 3—figure supplement 2).

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Gi/o-bound complexes have two conserved contacts: one between Gly^H5.24 and the TM7/H8 turn, and one between Tyr/Phe^H5.26 and TM6. In Gs-bound complexes, three distinct interactions are found: between Arg^H5.12 and TM3/ICL2, and between Leu^H5.26 and Arg^H5.17 and TM5 (Figure 3C,D). In Gi/o complexes, the equivalent Lys^H5.17 lies instead roughly parallel to TM6. Interestingly, Arg^H5.17 appears to consistently interact with residue 5.68 in class A GPCRs, and with Lys^5.64 in class B GPCRs (Figure 3E, Figure 3—figure supplement 2). We suggest that a tight interaction between Arg^H5.17 and TM5 might be one of the main determinants for the Gs-specific relocation of TM6.

Besides the canonical interaction with the α5 helix, our complex shows that ICL2 and ICL3 of the receptor are at contact distance to Gi (Figure 3—figure supplement 3A). In all analyzed structures, we found that ICL2 lies near αN/β1 and β2/β3 of Gα, while ICL3 is close to α4/β6 (Figure 3—figure supplements 3 and 4). Interestingly, ICL2 folds into an α-helical structure in all the class A receptors except rhodopsin (Figure 3—figure supplement 5).

ICL2 does not contribute to binding Gα in the structures of 5HT_1B-mini-Go and A_1AR-Gi. Nevertheless, the contact between Gα and ICL2 seems to discriminate between class A GPCRs –which interact via the αN/β1 loop– and class B GPCRs –which instead the region around β1 and β2/β3 (Figure 3—figure supplement 4).

ICL3, one of the most diverse regions in GPCRs, is often not completely resolved in the available structures (Figure 3—figure supplement 3D), either because it is too flexible or because it has been engineered to facilitate structural determination (Munk et al., 2019). Gi/o-coupled receptors use residues on TM5/ICL3/TM6 to contact the α4/β6 region, while Gs-coupled receptors mainly use TM5/ICL3 (Figure 3—figure supplement 4). This difference is due to the larger displacement of TM6 in Gs complexes. In ICL3, residue Tyr^G.S6.02, highly conserved in all G protein subtypes, engages the receptor in about half of all the complexes. This residue has been shown to be crucial for the stabilization of the rhodopsin-Gi complex (Sun et al., 2015).

Thus, our analysis suggests that ICL2 and ICL3 contribute to the binding interface between receptor and G protein, a feature that may be required to further stabilize the complex during nucleotide exchange.

In this work, we present the cryo-EM structure of the signaling complex between bovine rhodopsin and a Gi protein heterotrimer, stabilized using an antibody Fab fragment (Fab16) (Maeda et al., 2018). Overall, this structure agrees very well with existing complexes. In particular, we found that the binding mode of the G protein Ras domain –including the key C-terminal α5 helix– is virtually identical to our previously reported X-ray structure of the same receptor bound to a mini-Go protein (Tsai et al., 2018) (Figure 1—figure supplement 7B).

However, our EM map shows a density on the Gβ subunit that extends from H8 of the receptor, constituting the proximal segment of the C-terminus (residues G324 to T335) (Figure 2). One explanation of why the C-tail is observed in our density map and not in other structures may rely on the nature of the components used to reconstitute the reported GPCR complexes (Figure 1—figure supplement 8). Among those, a meaningful comparison may be done with the μ-opioid receptor complex (Koehl et al., 2018), which is bound to a shorter version of our antibody Fab16, but in which the C-terminus of the receptor was partially truncated. In the human rhodopsin complex (Kang et al., 2018), which contains the full length C-tail, a recombinant Gβγ was used. Thus, our bovine rhodopsin complex contains the unique combination of a full-length C-tail, Gβγ purified from bovine rod outer segments, and Fab16 that may have contributed to trap the transient interaction of the intrinsically disordered C-tail to Gβ. Remarkably, the recently published structure of the M1 muscarinic acetylcholine receptor in complex with G11 and scFv16 (Maeda et al., 2019) reveals part of the receptor C-tail bound to the G protein in essentially the same conformation as in our structure (Figure 2—figure supplement 4).

The Gβγ subunit engages with a wide range of effectors (Dupré et al., 2009; Khan et al., 2013), including direct interactions with GPCRs. For instance, these can associate prior to trafficking to the plasma membrane (Dupré et al., 2006) where they can remain pre-coupled (Galés et al., 2005). Also, according to a sequential fit model (Herrmann et al., 2004; Herrmann et al., 2006), activated rhodopsin binds to Gβγ assisting to position the Ras domain of Gα into proximity of its binding site to the receptor. This would facilitate folding of the C-terminal α5 helix of this subunit leading to nucleotide exchange (Dror et al., 2015; Flock et al., 2015; Kapoor et al., 2009). After dissociation of the G protein heterotrimer, the Gβ subunit recruits GRKs to the membrane, resulting in the phosphorylation of the receptor C-tail (Claing et al., 2002; Pao and Benovic, 2002; Pitcher et al., 1998) and binding of arrestin (Goodman et al., 1996). Interestingly, HEK cells lacking functional Gα subunits (but retaining the native Gβγ) are still able to recruit arrestin, although they fail to activate ERK and whole-cell responses (Grundmann et al., 2018).

Our findings provide a structural explanation for these roles of Gβγ in GPCR-mediated signaling. We suggest that the observed interaction between the central part of the receptor C-terminus and Gβγ is involved in localizing the G protein heterotrimer to the active receptor first. After dissociation of the Gα subunit, a transient Gβγ/receptor complex could provide the adequate molecular context to allow receptor phosphorylation by bringing the kinase close to the receptor C-tail (Ribas et al., 2007).

In other class A GPCRs, interactions with Gβγ have been also located in ICL3 (in M2 and M3) muscarinic receptors, involved in the phosphorylation of this loop (Wu et al., 1998) and potentially in ICL1 (in A₂AR and β₂AR) (García-Nafría et al., 2018a). In our complex, K6612.48 and K6712.49 in ICL1 are indeed close to D312 in Gβ (7 Å between Cα atoms); however, we do not observe defined density for the side chains in this region (Figure 1—figure supplement 6A). Interestingly, the complexes of the class B GPCRs calcitonin and GLP-1 feature an extended and tilted H8 resulting in extensive contacts with Gβγ that have not been observed in Class A receptors (García-Nafría et al., 2018a). H8 has also been shown to bind Gβγ in the class B PTH1 receptor (Mahon et al., 2006). Thus, it is likely that class A and class B GPCRs use distinct mechanisms to engage Gβγ.

The receptor-Gβγ interaction observed in our structure is compatible with proposed models of receptor-GRK recognition (Komolov et al., 2017; Sarnago et al., 2003). Thus, we suggest that the Gβγ subunit can ‘tag’ activated GPCRs and provide an interface for bringing different effectors (Gα subunits first, and GRKs later) near the cytoplasmic domains of GPCRs.

The availability of several GPCR-G protein complexes has greatly advanced our understanding on how receptors activate the Gα subunit (Dror et al., 2015; Flock et al., 2015). The binding interface of the receptor is partially formed by ICL2 and ICL3 (Chung et al., 2011; Glukhova et al., 2018; Sun et al., 2015). Accordingly, our EM map shows that these domains are in close proximity to G (Figure 3—figure supplement 3). In particular, we found that ICL2 is at contact distance to the αN helix, the αN/β1 and the β2/β3 Gα in most structures (Figure 3—figure supplements 3–5). While ICL2 contributes to the binding interface, there are no apparent conserved contacts among complexes (Figure 3—figure supplement 4). However, at the interface between receptor and the α5 helix of the G protein, in all Gi complexes Phe^H5.26 and Gly^H5.24 contact TM6 and TM7/H8 of the receptor, respectively. Instead, in all Gs complexes we find that Leu^H5.26, Arg^H5.17 and Arg^H5.12 contact TM5/ICL3, TM5, and TM3/ICL2, respectively. In a recent analysis (Glukhova et al., 2018), it was proposed that residues 5.68 (class A GPCRs) and 5.64 (class B GPCRs) are particularly important for Gs protein binding. We observe that residue 5.68 interacts with H5.16 in both Gi- and Gs-bound complexes, but only with Arg^H5.17 in all Gs complexes (Figure 3—figure supplement 2). While our analysis is limited by the number of available structures, we suggest that the conserved contacts identified between α5 and the receptors are structurally important anchoring points, but we cannot exclude that they might be relevant at the level of recognition between G protein subtypes.

In order to visualize snapshots of the signaling pathway that have not been observed yet, more work on the structural characterization of complexes is still needed, ideally using the same receptor and different transducers. Such complexes are of pivotal importance to decipher the structural basis of the GPCR-mediated signaling cascade.

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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Author details

1. Ching-Ju Tsai

   Division of Biology and Chemistry / Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen, Switzerland

   Contribution

   Formal analysis, Investigation, Visualization, Writing—original draft, Writing—review and editing

   Contributed equally with

   Jacopo Marino, Ricardo Adaixo and Filip Pamula

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8320-5009

2. Jacopo Marino

   Division of Biology and Chemistry / Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen, Switzerland

   Contribution

   Formal analysis, Funding acquisition, Investigation, Visualization, Writing—original draft, Writing—review and editing

   Contributed equally with

   Ching-Ju Tsai, Ricardo Adaixo and Filip Pamula

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7095-0800

3. Ricardo Adaixo

   Center for Cellular Imaging and NanAnalytics (C-CINA), Biozentrum, University of Basel, Basel, Switzerland

   Contribution

   Formal analysis, Investigation, Writing—review and editing

   Contributed equally with

   Ching-Ju Tsai, Jacopo Marino and Filip Pamula

   Competing interests

   No competing interests declared

4. Filip Pamula

   Division of Biology and Chemistry / Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen, Switzerland

   Contribution

   Formal analysis, Investigation, Writing—review and editing

   Contributed equally with

   Ching-Ju Tsai, Jacopo Marino and Ricardo Adaixo

   Competing interests

   No competing interests declared

5. Jonas Muehle

   Division of Biology and Chemistry / Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen, Switzerland

   Contribution

   Resources, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

6. Shoji Maeda

   Division of Biology and Chemistry / Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen, Switzerland

   Present address

   Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States

   Contribution

   Resources, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

7. Tilman Flock

   1. Division of Biology and Chemistry / Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen, Switzerland
   2. Department of Biology, ETH Zurich, Zürich, Switzerland

   Contribution

   Formal analysis, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

8. Nicholas MI Taylor

   Center for Cellular Imaging and NanAnalytics (C-CINA), Biozentrum, University of Basel, Basel, Switzerland

   Present address

   Novo Nordisk Foundation Center For Protein Research, University of Copenhagen, Denmark, Europe

   Contribution

   Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0761-4921

9. Inayatulla Mohammed

   Center for Cellular Imaging and NanAnalytics (C-CINA), Biozentrum, University of Basel, Basel, Switzerland

   Contribution

   Resources

   Competing interests

   No competing interests declared

10. Hugues Matile

    Pharma Research and Early Development, Therapeutic modalities, Roche Innovation Center Basel, Hoffmann-La Roche Ltd, Basel, Switzerland

    Contribution

    Resources, Investigation

    Competing interests

    Employee of Hoffmann-La Roche Ltd

11. Roger JP Dawson

    Pharma Research and Early Development, Therapeutic modalities, Roche Innovation Center Basel, Hoffmann-La Roche Ltd, Basel, Switzerland

    Contribution

    Resources, Investigation, Writing—review and editing

    Competing interests

    Employee of Hoffmann-La Roche Ltd

12. Xavier Deupi

    1. Division of Biology and Chemistry / Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen, Switzerland
    2. Condensed Matter Theory Group, Paul Scherrer Institute, Villigen, Switzerland

    Contribution

    Formal analysis, Supervision, Funding acquisition, Writing—original draft, Writing—review and editing

    For correspondence

    xavier.deupi@psi.ch

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-4572-9316

13. Henning Stahlberg

    Center for Cellular Imaging and NanAnalytics (C-CINA), Biozentrum, University of Basel, Basel, Switzerland

    Contribution

    Supervision, Funding acquisition, Writing—review and editing

    For correspondence

    Henning.Stahlberg@unibas.ch

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-1185-4592

14. Gebhard Schertler

    1. Division of Biology and Chemistry / Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen, Switzerland
    2. Department of Biology, ETH Zurich, Zürich, Switzerland

    Contribution

    Conceptualization, Supervision, Funding acquisition, Project administration, Writing—review and editing

    For correspondence

    gebhard.schertler@psi.ch

    Competing interests

    declares that he is a co-founder and scientific advisor of the company leadXpro AG and InterAx Biotech AG, and that he has been a member of the MAX IV Scientific Advisory Committee during the time when the research has been performed.

    "This ORCID iD identifies the author of this article:" 0000-0002-5846-6810

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