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Structure of the class D GPCR Ste2 dimer coupled to two G proteins

Nature volume 589, pages 148–153 (2021)Cite this article

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Abstract

G-protein-coupled receptors (GPCRs) are divided phylogenetically into six classes1,2, denoted A to F. More than 370 structures of vertebrate GPCRs (belonging to classes A, B, C and F) have been determined, leading to a substantial understanding of their function3. By contrast, there are no structures of class D GPCRs, which are found exclusively in fungi where they regulate survival and reproduction. Here we determine the structure of a class D GPCR, the Saccharomyces cerevisiae pheromone receptor Ste2, in an active state coupled to the heterotrimeric G protein Gpa1–Ste4–Ste18. Ste2 was purified as a homodimer coupled to two G proteins. The dimer interface of Ste2 is formed by the N terminus, the transmembrane helices H1, H2 and H7, and the first extracellular loop ECL1. We establish a class D1 generic residue numbering system (CD1) to enable comparisons with orthologues and with other GPCR classes. The structure of Ste2 bears similarities in overall topology to class A GPCRs, but the transmembrane helix H4 is shifted by more than 20 Å and the G-protein-binding site is a shallow groove rather than a cleft. The structure provides a template for the design of novel drugs to target fungal GPCRs, which could be used to treat numerous intractable fungal diseases4.

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Fig. 1: Overall cryo-EM reconstruction of the Ste2–G-protein heterotrimer complex.
Fig. 2: The Ste2 dimer interface.
Fig. 3: |α-Factor-binding site.
Fig. 4: G-protein-binding site.
Fig. 5: Comparison of Ste2 with mammalian GPCRs.

Data availability

The structure of the Ste2 dimer coupled to G proteins has been deposited in the Protein Data Bank (PDB) with accession code 7AD3, and the associated cryo-EM data has been deposited in the Electron Microscopy Data Bank (EMDB) with accession code EMDB-11720. The cryo-EM datasets eBIC Krios1, LMB Krios1 and LMB Krios2 are available from the Electron Microscopy Public Image Archive (EMPIAR; https://www.ebi.ac.uk/pdbe/emdb/empiar/) with the accession code EMPIAR-10550. There are no restrictions on data availability. Ste2 orthologue sequences and the generic residue numbering are available at https://gpcrdb.org/protein/ste2_yeast and the structure-based sequence alignments are available at https://gpcrdb.org/alignment/targetselection. Other structures discussed in this Article (with PDB codes 6G79, 3SN6, 6B3J, 7C7Q, 6D32, 6CMO and 6OFJ) are available at https://www.rcsb.org/.

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Acknowledgements

V.V. was funded by a Gates Cambridge Scholarship. Work in the C.G.T. laboratory was funded by a grant from the European Research Council (EMPSI 339995), Sosei Heptares and core funding from the Medical Research Council (MRC U105197215). Work in the N.V. laboratory was funded by grants from the National Institutes of Health (R01-GM117923, R01-GM097261). Work in the D.E.G. laboratory was funded by Independent Research Fund Denmark | Natural Sciences (8021-00173B), Lundbeck Foundation (R163-2013-16327) and Novo Nordisk Foundation (NNF18OC0031226). D.E.G. is a member of the Integrative Structural Biology at the University of Copenhagen (ISBUC) cluster. We thank T. Nakane, P. Emsley, A. G. W. Leslie, S. Scheres, E. Miller, E. R. S. Kunji, P. Edwards, T. Warne and V. Chandrasekaran for discussions; and S. McLaughlin and C. Johnson from the Laboratory of Molecular Biology (LMB) biophysics facility, G. Cannone and G. Sharov from the LMB EM facility and J. Grimmett and T. Darling from the LMB scientific computing facility for technical support.

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Authors and Affiliations

  1. MRC Laboratory of Molecular Biology, Cambridge, UK

    Vaithish Velazhahan, Yang Lee & Christopher G. Tate

  2. Department of Computational and Quantitative Medicine, Beckman Research Institute of the City of Hope, Duarte, CA, USA

    Ning Ma & Nagarajan Vaidehi

  3. Department of Drug Design and Pharmacology, Universitetsparken 2, Copenhagen, Denmark

    Gáspár Pándy-Szekeres, Albert J. Kooistra & David E. Gloriam

  4. Medicinal Chemistry Research Group, Research Center for Natural Sciences, Budapest, Hungary

    Gáspár Pándy-Szekeres

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  1. Vaithish Velazhahan
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  8. Christopher G. Tate
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Contributions

V.V. performed receptor and G-protein engineering, expression, purification, complex formation, biophysical characterization, preparation of cryo-EM grids, cryo-EM data collection, data processing, structure determination and model building. N.M. performed the molecular dynamics simulations, and N.M. and N.V. analysed the molecular dynamics simulation trajectories. G.P.-S. developed the generic residue numbers and GPCRdb resources. A.J.K. analysed sequence conservation, structural mechanisms and co-supervised G.P.-S. Y.L. advised on cryo-EM data collection, data processing and model building. D.E.G. supervised G.P.-S. and A.J.K. and contributed to the design of residue numbering and GPCRdb resource. V.V. and C.G.T. carried out structure analysis and manuscript preparation. C.G.T. managed the overall project. The manuscript was written by V.V. and C.G.T., and included contributions from all authors.

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Correspondence to Christopher G. Tate.

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Competing interests

C.G.T. is a shareholder in, consultant to, and member of the scientific advisory board of Sosei Heptares, who also partly funded this work.

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Extended data figures and tables

Extended Data Fig. 1 Purification, formation and biophysical characterization of the Ste2-G-protein heterotrimer complex.

a, FSEC trace of insect cells expressing wild-type Ste2-TEV-eGFP-His10 solubilized in different detergents. Ste2 migrated as a dimer in all tested detergents with an apparent molecular weight of around 380 kDa. The molecular weight standards used for calibration of the column are shown for reference. Results are representative of three independent experiments. b, SEC profiles of Ste2 and SDS–PAGE analysis of the eluted fractions. c, SEC profiles of Ste2–G-protein heterotrimer complex and SDS–PAGE analysis of the eluted fractions from the complex formation reaction (see Methods). d, SDS–PAGE analysis of the pooled and concentrated Ste2–G-protein complex along with the individual components used to form the complex. Experiments in b–d are representative of three independent purifications. e, Complex stability as analysed by SEC after incubation of the purified complex on ice for up to 15 days. This experiment was carried out once. f, Thermal stability as analysed by nanoscale differential scanning fluorimetry, which measures changes in the intrinsic fluorescence of the protein when subjected to thermal denaturation. The melting temperatures (Tm) of the Ste2–G-protein complex and the Ste2 dimer were calculated to be 51.2 °C and 55.0 °C, respectively. This experiment was performed once in two replicates. g, Representative 2D class averages of Ste2 imaged on a Titan Krios with the GIF Quantum K2 detector and a Volta Phase Plate56 (VPP). All classes are consistent with a dimer and no monomer classes were observed. h, SEC–MALS analysis of the Ste2–mini-G-protein heterotrimer complex, Ste2–wild-type G-protein heterotrimer complex, Ste2 dimer and mini-G-protein heterotrimer complex. A large change in refractive index that occurs at the end of the column volume due to the elution of salts and/or detergents present in the solvent is indicated as the solvent peak. Where indicated, chromatograms show signal from UV absorbance, light scattering and refractive index. The traces were normalized to the peak maxima. Calculated molar masses are depicted as traces. The molar masses of the protein components across the peak and the theoretical molar masses are indicated. SDS–PAGE analysis of the Ste2–mini-G-protein heterotrimer and Ste2–wild-type G-protein heterotrimer complexes are shown. The results are representative of three independent experiments, except the experiment with the Ste2 dimer, which was performed once. For gel source data (b–d, h), see Supplementary Fig. 1.

Extended Data Fig. 2 Cryo-EM single-particle reconstruction of the Ste2–G-protein heterotrimer complex.

a, A representative micrograph (defocus −2.0 μm) from the eBIC Krios1 dataset. b, Representative 2D class averages of the Ste2–G-protein heterotrimer complex determined using the initial set of particles from eBIC Krios1 dataset following 3D classification. Reconstructions with the detergent micelle are juxtaposed to indicate relative orientations. c, A representative 2D class average is shown from the final set of particles (eBIC Krios1 dataset) used to determine the model. Whereas one G protein is well-resolved, the second heterotrimeric G protein manifests as a poorly resolved density in the reference-free 2D averages (circled). d, The poorly resolved G-protein heterotrimer (orange circle) becomes apparent at lower contour levels and certain secondary structural features become visible when the map is low-pass-filtered to 8 Å. The contact between mini-Gpa1B and Ste4A of the adjacent mini-Gpa1A heterotrimer is indicated (pink circle) e, g, FSC curves of the reconstructions map 1 (e) and map 2 (g) (black lines) shows an overall resolution of 3.5 Å and 3.3 Å, respectively, using the gold standard FSC of 0.143. The directional 3D-FSC curves computed from the two half-maps are shown in colour. h, Local resolution estimation of the Ste2 dimer portion from map 2 as calculated by RELION. f, i, Local resolution estimation of the Ste2–G-protein heterotrimer complex from map 1 (f) and map 2 (i) as calculated by RELION. Map 2 shows a higher local resolution in the Ste2 dimer, but the resolution is limited in the G-protein regions.

Extended Data Fig. 3 Flow chart of single-particle cryo-EM data processing.

Micrographs were collected from three independent sessions (between 24-h and 72-h long) on a Titan Krios with GIF Quantum K2. Each dataset was corrected separately for drift, beam-induced motion and radiation damage. After estimation of CTF parameters, particles were picked using a Gaussian blob and subjected to two rounds of 3D classification using an ab initio model as reference. The number of final particles from each round of 3D classification is shown. A representative 3D classification scheme (round 2) from the eBIC Krios1 dataset is shown. The initial set of best particles from the eBIC Krios1 dataset was refined and corrected for beam-tilt. Per-particle CTF estimation and Bayesian polishing was performed, detergent micelle signal was subtracted, and a 3D classification without alignment was performed. A model (map 1) with 96,611 particles was refined and achieved a global resolution of 3.5 Å. In parallel, the final set of particles from LMB Krios1 and LMB Krios2 were refined and corrected for beam-tilt, which was followed by per-particle CTF estimation and Bayesian polishing. The initial set of particles from eBIC Krios1 was combined with the final set of particles from LMB Krios1 and LMB Krios2. The combined set of particles was refined, anisotropic magnification correction was performed, and the particles were subjected to a 3D classification without alignment after subtraction of the signal from the detergent micelle and the G protein. After restoring the G-protein signal, a set of 131,266 particles was refined to a global resolution of 3.3 Å (map 2) with improved features in the Ste2 dimer region. Resolution of the models after refinements was calculated with the gold-standard FSC of 0.143 in RELION.

Extended Data Fig. 4 Cryo-EM map quality of the Ste2–mini-Gpa1–Ste4–Ste18 heterotrimer complex and model validation.

Unless stated otherwise, densities from map 1 were visualized using UCSF ChimeraX35 (contour level 0.03) and encompass a carve radius of 2 Å around the indicated region. a, Densities of transmembrane helices of Ste2A (contour level 0.045), α-factorB, mini-Gpa1B, dimer interface between Ste2A and Ste2B, intracellular loops ICL1–3 and extracellular loops ECL1–3 are shown as a mesh. Densities of αN and α4–β6 are shown as representative densities from mini-Gpa1A. Densities of Ste4 and Ste18 (contour 0.025) are shown as a mesh at a contour level of 0.01 and a carve radius of 2 Å. b, Two N-acetylglucosamine molecules were modelled attached to Asn25 of SteA and Ste2B and they contribute to the dimer interface (contour level 0.01 from map 2). Asn25 is a well-characterized glycosylation site in Ste217. c, Densities for six ordered putative CHS molecules surrounding the dimer interface were visible and were modelled (contour level 0.01). Several other detergent or lipid acyl-chain densities were visible in the map before detergent micelle signal subtraction, but were left unmodelled. d, FSC of the refined model versus the map (green curve) and FSCwork/FSCfree validation curves (orange and blue curves, respectively).

Extended Data Fig. 5 Comparison of Ste2 with other classes of GPCRs.

Thirty-nine receptors in the active state were aligned using GESAMT73 and then prototypical members of different classes were compared with Ste2. Both GESAMT73 and TM-align72 provided similar results. In all panels, Ste2 is shown in rainbow colouration and is compared with: a, β2-adrenoceptor (class A; PDB ID: 3SN6); b, glucagon-like peptide receptor 1 (class B; PDB ID: 6B3J); c, GABAB receptor (class C, PDB ID: 7C7Q); d, smoothened (class F; PDB ID: 6D32).

Extended Data Fig. 6 Amino acid conservation of Ste2 and the CD1 numbering system.

a, The size of the letter is related to the degree of conservation from the aligned Ste2 orthologues (395 sequences). The CD1 numbering system is shown alongside the positions of the defining residue and Ballesteros–Weinstein number for class A GPCRs. Generic residue numbers use the GPCRdb scheme, which uses the same reference positions as the Ballesteros–Weinstein scheme but corrects offsets due to helix bulges or constrictions. Two structurally important motifs in class A GPCRs (DRY and NPXXY) are depicted. b, The amino acid sequence of S. cerevisiae Ste2 is shown (coloured boxes) in relation to secondary structural elements. The CD1 number (for example, 5x50) is given adjacent to the amino acid residue number. Numbers in grey boxes represent the sequence conservation of the S. cerevisiae Ste2 residue at that position among Ste2 orthologues from 395 unique sequences. The light blue colour on the residue sequence numbers indicates residues predicted to be positioned in the lipid bilayer on the basis of structural superposition with the class B receptor GLP1R (PDB ID: 6B3J).

Extended Data Fig. 7 Amino acid residues forming the Ste2 dimer interface.

Contacts are shown as coloured squares with a digit representing the number of van der Waals interactions (≤3.9 Å) and hydrogen bonds.

Extended Data Fig. 8 Molecular dynamics simulations of the Ste2 dimer in the absence of G proteins and ligands.

a, Groove in the H1–H1 dimer interface. b, Heat map of the percentage of molecular dynamics snapshots that show residue contacts in the dimer interface in the EM structure and in the representative structure from molecular dynamics simulations in the absence of the ligands and G proteins. c, Top row, histograms of the standard deviation among 25 simulation runs (total 1.4 μs) for Ste2 dimer with G protein and α-factor. Bottom row, histograms of the standard deviation in Ste2A and Ste2B interface residue contacts among 5 simulation runs (total 5 μs) of the Ste2 dimer without G protein. d, Convergence of the five 1-μs simulations was tested by calculating the dot product among the top 5 weighted principal components (PCs) for the 5 simulations of Ste2 dimer without G protein or α-factor. The top weighted principal components (PC1 to PC5) from each run all show strong overlap (red squares) with at least one of the PCs from other runs. e, Cumulative variance of PCs show that the top five PCs occupy more than 90% of the populations, and therefore they are sufficient to describe the motion of the complex.

Extended Data Fig. 9 Structural features of Ste2 and comparison with mammalian GPCRs.

a–c, Alignment of GPCRs was performed by GESAMT73 (CCP4 suite of programs) in conjunction with 39 other G-protein- or arrestin-coupled GPCR structures (Fig. 5a). Ste2 is depicted in rainbow colouration and the adenosine A2A receptor (A2AR) is in grey. a, DRY motif showing the stacking (van der Waals contacts, yellow dashed lines) of the highly conserved Arg3x50 residue with PheH5.23 of the α-subunit of Gs. b, The Pro5x50-Ile3x40-Phe6x44 motif provides a conserved packing of residues in the active state at the core of class A receptors. A similar role could be played by Leu5x35 and Val3x40 in Ste2. c, The NPXXY motif in class A GPCRs aligns with a conserved region in Ste2 receptors (see also Extended Data Fig. 6a), but Pro7x50 in Ste2 is not associated with a kink in H7 as it is in class A GPCRs. d, Conformational changes in the transmembrane α-helices observed during molecular dynamics simulations of the dimer without the G proteins or the ligands. e, Alignment of the active-state Ste2 (rainbow colouration) with the Ste2 conformation (grey) reached after molecular dynamics simulations in the absence of G protein and ligand. Gpa1 would not be able to couple to this new conformation owing to clashes with atoms in H2 (Ile80), H3 (Phe148, Gln149, Lys151, Val152, Thr155) and ICL2 (Gly156, Asp157, Asn158, Phe159). Clashes are defined as atoms (grey spheres) within 2 Å of atoms in Gpa1 coupled to the active state of Ste2. f, The position of the α-helical domain in Gpa1 distal to the dimer interface can be inferred from comparison with the structure of Gi (yellow) coupled to rhodopsin (PDB ID: 6CMO) after alignment with mini-Gpa1 (purple). The position in Gpa1 of the deletion of the α-helical domain (which also includes the ubiquitination domain) is indicated by the amino acid residues immediately before and after the deletion being shown as spheres. g, Superposition of Go-coupled serotonin 5-HT1B receptor (yellow) with Ste2A (blue). The Gpa1 heterotrimer is rotated by 58° (angle between the centre of masses of Gβ subunit of Go heterotrimer and Ste4 of Gpa1 heterotrimer) perpendicular to the membrane plane, which creates sufficient space for two G-protein heterotrimers to simultaneously couple to the Ste2 dimer. h, Views of the intracellular surfaces of dimers from class D (Ste2), class A (rhodopsin; PDB ID: 6OFJ) and class C (GABAB receptor; PDB ID: 7C7Q). All receptors are in rainbow colouration.

Extended Data Table 1 Cryo-EM data collection and refinement statistics
Full size table

Supplementary information

Supplementary Figure 1

Uncropped SDS-PAGE gels. The Coomassie Blue-stained SDS PAGE gels used to generate the cropped panels in Extended Data Fig. 1 are depicted in full.

Reporting Summary

Peer Review File

Supplementary Table 1

Dimer interface. Previous published mutations of amino acid residues at the Ste2 dimer interface are listed, with associated changes in ligand binding and receptor signalling, and the literature source.

Supplementary Table 2

Ste2 conservation and source organisms. The conservation of amino acid residues in the Ste2 receptor is shown along with the list of all organisms containing Ste2 orthologues used in the analysis.

Supplementary Table 3

Ste2 mutations in the orthosteric binding site. Previous published mutations of amino acid residues at the Ste2 orthosteric binding site are listed, with associated changes in ligand binding and receptor signalling, and the literature source.

Supplementary Table 4

Mutations in α-factor. Previous published alterations of amino acid residues in α-factor are listed, with associated changes in ligand binding and receptor signalling, and the literature source.

Supplementary Table 5

G-protein mutations. Previous published mutations of amino acid residues in Gpa1 found at the receptor-G protein interface are listed, with associated changes in Ste2 signalling, and the literature source.

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Velazhahan, V., Ma, N., Pándy-Szekeres, G. et al. Structure of the class D GPCR Ste2 dimer coupled to two G proteins. Nature 589, 148–153 (2021). https://doi.org/10.1038/s41586-020-2994-1

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