Cryo-EM structure of the serotonin 5-HT1B receptor coupled to heterotrimeric Go

Abstract

G-protein-coupled receptors (GPCRs) form the largest family of receptors encoded by the human genome (around 800 genes). They transduce signals by coupling to a small number of heterotrimeric G proteins (16 genes encoding different α-subunits). Each human cell contains several GPCRs and G proteins. The structural determinants of coupling of G_s to four different GPCRs have been elucidated^1-4, but the molecular details of how the other G-protein classes couple to GPCRs are unknown. Here we present the cryo-electron microscopy structure of the serotonin 5-HT_1B receptor (5-HT_1BR) bound to the agonist donitriptan and coupled to an engineered G_o heterotrimer. In this complex, 5-HT_1BR is in an active state; the intracellular domain of the receptor is in a similar conformation to that observed for the β₂-adrenoceptor (β₂AR) ³ or the adenosine A_2A receptor (A_2AR) ¹ in complex with G_s. In contrast to the complexes with G_s, the gap between the receptor and the Gβ-subunit in the G_o-5-HT_1BR complex precludes molecular contacts, and the interface between the Gα-subunit of G_o and the receptor is considerably smaller. These differences are likely to be caused by the differences in the interactions with the C terminus of the G_o α-subunit. The molecular variations between the interfaces of G_o and G_s in complex with GPCRs may contribute substantially to both the specificity of coupling and the kinetics of signalling.

Extended Data Figure 1. Cryo-EM single particle reconstruction of the 5-HT_1BR–G_O complex structure.

a, Representative micrograph (magnification 75,000x, defocus -0.6 μm) of the 5-HT_1BR–G_O complex collected using a Titan Krios with the Falcon III detector and Volta phase plate. b, Representative 2D class averages of the 5-HT_1BR–G_O complex. c, FSC curve of the final reconstruction showing an overall resolution of 3.8 Å using the gold-standard FSC of 0.143. Both masked and unmasked FSC curves are shown to highlight the lack of masking artefacts. d, Final reconstruction coloured by subunit showing a zoom on the weak density for ICL3. The zoomed region corresponds to a map sharpened with B = -50 to remove noise from lower density levels. e, Local resolution estimation of the 5-HT_1BR map as calculated by Resmap.

Extended Data Figure 2. Cryo-EM map quality and model validation.

a, transmembrane helices of 5-HT_1BR; b, the α5 helix of G_O; c, donitriptan and the neighbouring side chains in the orthosteric binding site; d, Fourier shell correlation of the refined model versus the map (green curve) and FSC_work/FSC_test validation curves (blue and red curves, respectively).

Extended Data Figure 3. Flow-chart of data processing.

Micrographs were collected during nine sessions on the Titan Krios (either 24 h or 48 h) and each session was processed independently. The number of images and particles for one 48 h session is indicated on the flowchart as a guide. At the bottom of the figure, the final number of particles is shown. Each dataset was corrected separately for drift, beam induced motion and radiation damage. After CTF estimation, particles were picked using a Gaussian blob and submitted to either one or two rounds of reference-free 2D classification (see Methods). A 3D classification was performed on the selected particles using an ab initio model generated from ten thousand particles. Classification was performed in parallel in three and four classes. The models with best features were refined on their own; if there were two classes of similar high quality, these were then re-refined together (the resolution of the models refers to that after refinement and calculation of gold-standard FSC=0.143). The set of particles that obtained the best map quality and resolution were saved and merged with the best particles from other datasets. A final model with 730,118 particles was refined and achieved a global resolution of 3.78 Å.

Extended Data Figure 4. Modelling quality of the 5-HT_1BR structure.

a, Amino acid sequence of 5-HT_1BR construct used in the cryo-EM structure determination. Residues are colored according to how they have been modelled: black, good density allows the side chain to be modelled; red, limited density for the side chain present and therefore the side chain has been truncated to Cβ; blue, no density observed and therefore the residue was not modelled. Regions highlighted in grey represent the transmembrane α-helices and amphipathic helix 8 is highlighted in yellow. b, Model of 5-HT_1BR showing the Cα positions of amino acid residues with poor density (spheres) and regions unmodelled (dotted lines).

Extended Data Figure 5. Superposition of donitriptan, adrenaline and adenosine.

5-HT_1BR, β₂AR³ and A_2AR¹ were superimposed (Pymol) over the whole of the receptor and the ligands coloured accordingly: green, donitriptan; pink, adrenaline; blue, adenosine.

Extended Data Figure 6. Comparison of the amino acid sequences of the α subunits of G_O and G_S.

Diamonds above the sequences identify the amino acid residues in Gα_s where the side chains that make atomic contacts to residues in either β₂AR (β2 con) or A_2AR (2A con). Ovals above the sequences identify the amino acid residues in Gα_s where only the main chain atoms make contacts to the receptor. Secondary structural elements are indicated as grey bars and the CGN system of numbering is shown.

Extended Data Figure 7. Similarity of Gα structures and the difference poses of the α5 helices in Gα_O and Gα_S coupled to receptors.

a, The structures of the α subunits coupled to 5-HT_1BR, β₂AR³ and A_2AR¹ were superimposed over the whole of their sequence in Pymol; blue, Gα_O coupled to 5-HT_1BR; green, Gα_S coupled to A_2AR; Gα_S coupled to β₂AR. b, 5-HT_1BR (blue), β₂AR³ (green) and A_2AR¹ (red) were superimposed based on H3, H5 and H6. Two different views are shown with the red arrows indicating differences in orientation of Gα_S and Gα_O.

Extended Data Figure 8. Alignment of the amino acid sequences of G_O and G_i α subunits.

Sequences in grey correspond to the α-helical region that do not make contact to GPCRs and was deleted during the construction of mini-G_O. Secondary structural elements are depicted as grey bars with the CGN system shown to aid comparisons. Amino acids are highlighted as follows: pink, stabilizing residues required to generate mini-G_O; yellow; residues in Gα_O that are different from residues conserved in all three Gα_i sequences; blue, residues that are non-conserved in Gα_i sequences. # represents the affinity tag on mini-Go used for purification (MGHHHHHHENLYFQG).

Extended Data Figure 9. Comparison between the α5 helices of G_S and G_O.

The α5 helices in the cryo-EM structures of A_2AR-G_S (carbon, green) and 5-HT_1BR-G_O (carbon, light blue) were aligned (Pymol) along their whole sequence and displayed in different poses: a, cartoon depiction; b, G_S (green spheres), G_O, (blue sticks); c, G_O (blue spheres), G_S, (green sticks).

Figure 1. Overall cryo-EM reconstruction of the 5-HT_1BR–G_O heterotrimer complex.

The density for the cryo-EM map (sharpened with a B factor of -200) is coloured according to the subunit. The inset shows the orthosteric binding pocket in 5-HT_1BR (light blue) with donitriptan depicted as sticks (green, carbon) and its density in the cryo-EM map. The lower panel shows a superposition of ergotamine-bound 5-HT_1BR (pale grey, PDB ID 4IAR) and donitriptan-bound 5-HT_1BR (pale blue). Donitriptan (green, carbon) and ergotamine (orange, carbon) are depicted as sticks.

Figure 2. G_O-coupled 5-HT_1BR is in an active conformation.

a-c, Superposition of G protein-bound receptors: 5-HT_1BR (blue), A_2AR (red)¹ and β₂AR (green)³ based on H3, H5 and H6. Key amino acid residues involved in receptor activation are displayed as sticks. d-f, Superposition of G_O-coupled 5-HT_1BR (blue) and the active-intermediate state of 5-HT_1BR bound to ergotamine (pale grey), based on alignment of the whole receptor. Conformational changes involved in receptor activation are highlighted (red arrows) and key residues are shown as sticks with the density from the cryo-EM map (mesh). a and d, view parallel to the membrane plane; b and e, enlarged view of the conserved core of the receptors; c and f, view from the cytoplasmic face of the membrane.

Figure 3. G_O coupling to the 5-HT_1BR.

a, C-terminal end of Gα_O (yellow sticks) inserted into the cytoplasmic cleft of 5-HT_1BR (blue cartoon). Cryo-EM density is depicted as a mesh. b, Superposition of 5-HT_1BR (blue), A_2AR (red)¹ and β₂AR (green)³ based on alignment of H3, H5 and H6. The different poses of the C-termini of G_S and G_O coupled to the respective receptors are shown. c, Amino acid residues in 5-HT_1BR, A_2AR and β₂AR that make contact to the respective Gα subunits they are coupled to are shown in colours that reflect biophysical properties of the residues; green, hydrophobic; yellow, hydrophilic, red, acidic; blue, basic. Residues coloured in white do not make contact to the relevant Gα. The amino acid alignment was created in GPCRdb and secondary structural elements and the Ballesteros-Weinstein numbers are depicted. d, Snake plot of 5-HT_1BR created in GPCRdb with residues making contact to Gα_O coloured according to their biophysical properties. Regions in grey were disordered in the cryo-EM map. e, Cartoon of secondary structural elements in Gα_O and amino acid residues that make contact to 5-HT_1BR are depicted and coloured according to their biophysical properties.

Figure 4. Comparison of G_S coupling vs G_O coupling.

a, Cartoon of β₂AR (green) coupled to G_S (PDB ID 3SN6). The α-helical domain of the Gα has been removed for clarity. b-d, surface rendered views of the interface between a receptor and G protein: b, β₂AR (green) and G_S; c, A_2AR¹ (red) and G_S; d, 5-HT_1BR (blue) and G_O.