Structure of an insect gustatory receptor

SUMMARY Gustatory Receptors (GRs) are critical for insect chemosensation and are potential targets for controlling pests and disease vectors. However, GR structures have not been experimentally determined. We present structures of Bombyx mori Gr9 (BmGr9), a fructose-gated cation channel, in agonist-free and fructose-bound states. BmGr9 forms a tetramer similar to distantly related insect Olfactory Receptors (ORs). Upon fructose binding, BmGr9’s ion channel gate opens through helix S7b movements. In contrast to ORs, BmGR9’s ligand-binding pocket, shaped by a kinked helix S4 and a shorter extracellular S3-S4 loop, is larger and solvent accessible in both agonist-free and fructose-bound states. Also unlike ORs, fructose binding by BmGr9 involves helix S5 and a binding pocket lined with aromatic and polar residues. Structure-based sequence alignments reveal distinct patterns of ligand-binding pocket residue conservation in GR subfamilies associated with distinct ligand classes. These data provide insight into the molecular basis of GR ligand specificity and function.

(B) Sequence coverage for the three sequence alignments mapped onto the sequence (top) and structure (bo om) of the representa ve member, as follows: structure-driven alignment of 1895 GR sequences (includes 41 OR sequences) mapped to BmGr9; structure-driven alignment of 3885 OR sequences mapped to MhOr5; and previously published alignment of 176 ORCO sequences 24 mapped to AbOrco.
(C) Sequence covaria on analysis using the structure-driven alignment of 1854 GR sequences mapped to BmGr9.Intrasubunit structural contacts in BmGr9 are in orange (8 Å cutoff).The 28 evolu onarily coupled residue pairs above the 90% confidence threshold are indicated by black dots.(B) All GR and OR structures have fenestra ons between pore helices in the membrane plane.For each available GR or OR structure, the transparent surface and cartoon representa on of helix S7b from two adjacent subunits and the loop preceding helix S7b for the yellow subunit are illustrated.For the two BmGr9 structures, the modeled pore-penetra ng lipid is shown in black s cks; for the other structures, the fenestra on is marked by a black star.(E) Comparison of the central ion pore of the agonist-free structures of BmGr9 (le ), AbOrco (middle; PDB ID: 6C70), and MhOr5 (right; PDB ID: 7LIC).Helix S7b from two opposing protein subunits are shown in cartoon representa on, and the sidechains of residues that line the pore walls are shown in s cks and labeled.
(F) Sequence logos of the helix S7b posi ons (grey box) of alignments of 1854 insect GR sequences, 176 ORCO sequences, and 3885 insect OR sequences, with the reference sequences of BmGr9, AbOrco, and MhOr5, respec vely, indicated below the logo.Residues lining the central pore are marked with arrowheads colored orange (agonist-free structure) or blue (fructose-bound structure) or both colors for sidechains that are pore-lining in both structures.(D) Distance difference matrix of C-to-C distances for agonist-free versus fructose-bound BmGr9.The breaks in the axes mark missing residues 230-283.Thick black lines on the four axes mark the extracellular loops, and grey lines mark the helix boundaries.Black boxes mark the rela ve distances between the three pocket-forming regions of BmGr9, highligh ng that the extracellular regions generally move closer together upon fructose binding.(E) Zoomed-in views of the predicted ligand-binding pockets from the right-hand subunit of panels A-D to highlight rela ve size of the pockets across (sub)families.Some stray surfaces not corresponding to the pocket volumes were removed for clarity.

Figure S2 .
Figure S2.Cryo-EM processing procedure for the agonist-free BmGr9 structure Processing scheme for classifica on and refinement of the agonist-free BmGr9 map.The locally filtered map used for the final reconstruc on is represented with dust hidden for clarity.

Figure S3 .
Figure S3.Cryo-EM data analysis for the agonist-free BmGr9 structure (A) Representa ve micrograph of BmGr9 embedded in vitreous ice (scale bar = 250 Å), low pass filtered for clarity.(B) Selected 2D class averages of BmGr9.(C) Reconstruc on of BmGr9 filtered and colored by local resolu on.(D) Gold-standard Fourier shell correla on (FSC) curves from cryoSPARC.(E)Viewing direc on distribu on plot.

Figure S4 .
Figure S4.Cryo-EM processing procedure for the fructose-bound BmGr9 structure Processing scheme for classifica on and refinement of the fructose-bound BmGr9 map.The locally filtered map used for the final reconstruc on is represented with dust hidden for clarity.

Figure S5 .
Figure S5.Cryo-EM data analysis for the fructose-bound BmGr9 structure (A) Representa ve micrograph of BmGr9 in the presence of fructose embedded in vitreous ice (scale bar = 250 Å), low pass filtered for clarity.(B) Selected 2D class averages of fructose-bound BmGr9.(C) Reconstruc on of fructose-bound BmGr9 filtered and colored by local resolu on.(D) Gold-standard Fourier shell correla on (FSC) curves from cryoSPARC.(E)Viewing direc on distribu on plot.

Figure S6 .
Figure S6.Structural and sequence data suppor ng secondary structure and topology analyses (A) Cryo-EM densi es of individual helices for the agonist-free (le , orange) and fructose-bound (right, blue) BmGr9 structures.Protein is shown in s ck representa on with density contoured to 3.5σ and 4.5σ for the agonist-free and fructose-bound maps, respec vely.The helices (and S0-S1 -hairpin) are labeled below each helix from N to C terminus.

(
D) The 28 evolu onarily coupled residues pairs mapped onto the BmGr9 structure as black C-to-C bonds.Two opposing subunits are shown in transparent cartoon representa on colored in blue-to-red rainbow from N to C terminus.Nearly all top coupled pairs are in the intracellular anchor domain.Noteworthy structural features highlighted in Figure2Aare marked with black stars.

Figure S7 .
Figure S7.Density for a lipid penetra ng the ion pore is observed in other GR and OR structures (A) Structure of agonist-free BmGr9 illustra ng the posi on of the transmembrane fenestra on between the blue and yellow subunits with the bound pore-penetra ng lipid (black s cks).The boxed region is shown in B for this structure and other available structures of GRs and ORs.
(C-E) Transmembrane cross-sec ons of BrGr9 and MhOr5 structures viewed from the extracellular side of the membrane.Surfaces shown in black are densi es in the respec ve cryo-EM map corresponding to poten al pore-penetra ng lipids.The fructose-bound BmGr9 structure and map contoured at 3.5σ are shown in (C); the eugenol-bound MhOr5 structure (PDB ID: 7LID) and map (EMDB ID: 23374) contoured at 7.5σ in (D); and the DEET-bound MhOr5 (PDB ID: 7LIG) and map (EMDB ID: 23375) contoured at 7.5σ in (E).Visible helices of the yellow subunit are labeled.

Figure S8 .
Figure S8.Comparisons of pore between BmGr9 and AbOrco and MhOr5(A) The quadrivial pore of BmGr9 in the agonist-free (top) and fructose-bound structures illustrated as a surface colored according to its diameter.The cartoon representa on of two opposing protein subunits is included, and black lines make the membrane boundaries.(B-D)Electrosta cs surface representa on of BmGr9 in the agonist-free (le ) and fructose-bound (right) structures, in three cross-sec ons as indicated by the cartoon representa ons and planes on the le : (B) Ver cal cross-sec ons through the ion pore.Black lines mark the membrane boundaries, and arrows point to the hydrophobic lateral fenestra ons filled with the pore-penetra ng lipids and the lateral conduits of the ion pore.(C) Horizontal cross-sec ons through the hydrophobic fenestra ons.(D) Horizontal crosssec ons through the lateral conduits.The electrosta cs poten als were calculated using APBS in PyMOL and colored as a range from -20 kcal/mol (red) to 20 kcal/mol (blue).

Figure S9 .
Figure S9.Conforma onal changes in and fructose docking into the BmGr9 ligand-binding pocket (A-B) Electrosta cs surface representa on of agonist-free (A) and fructose-bound (B) BmGr9, sliced through the two ligand-binding pockets of opposing subunits in the tetramer.The two pockets are indicated with black arrows.The electrosta cs poten als were calculated using APBS in PyMOL and colored as a range from -20 kcal/mol (red) to 20 kcal/mol (blue).(C) Cryo-EM density for the fructose-bound map contoured at 4.5σ around the fructose-binding site, with nearby sidechains shown as s cks.The five lowest-energy poses of docked -D-fructopyranose are illustrated in grey s cks, and the -D-fructopyranose pose a er real-space refinement of the lowest-energy pose against the cryo-EM density is shown in black s cks.

Figure S10 .
Figure S10.Posterior probability branch supports for the maximum likelihood phylogene c tree of insect GR sequencesA representa on of the phylogene c tree in Figure6Ashowing aBayes support values for each branch.Terminal branches are shown in grey.

Figure S11 .
Figure S11.Annotated phylogene c tree of insect GR sequencesMaximum likelihood phylogene c tree of 1895 aligned insect GR sequences.Black dots indicate posi ons of D. melanogaster GRs on the tree.The alignment included 41 OR sequences, marking the branch point of the OR family within the more ancestral GR sequences (orange).The three subfamilies analyzed in this work are highlighted: Gr5a subfamily in green, Gr63a subfamily in red, and Gr43a subfamily in purple.

Figure S12 .
Figure S12.Side views of predicted ligand-binding pockets in representa ve GR subfamily members (A-E) Cartoon representa on of two opposing subunits from representa ve subfamily members viewed from the membrane plane, with surface representa on of the atoms forming the predicted ligand-binding pockets to illustrate the rela ve loca on and size of the binding pockets.The cartoons and surfaces are colored by ConSurf conserva on score based on the sequence alignment of the corresponding GR subfamily.As in Figure 6, the following subunits and corresponding protein (sub)families are illustrated: (A) Gr43a subfamily (74 sequences) on the agonist-free BmGr9 structure; (B) Gr5a subfamily (251 sequences) on the AlphaFold2 model of Gr5a (UniProt ID: Q9W497); (C) Gr63a subfamily (107 sequences) on the AlphaFold2 model of Gr63a (UniProt ID: Q9VZL7); and (D) the OR family (3885 sequences) on the agonist-free MhOr5 structure (PDB ID: 7LIC).For the AlphaFold2 models, tetramers were built for visualiza on purposes by superposi on of four subunit models on the BmGr9 tetramer.

Figure S13 .
Figure S13.Distribu on of loop lengths across GR subfamilies, the GR family and the OR family (A) Violin plots depict the distribu on of lengths for each loop connec ng two adjacent helices as indicated in each plot.Each plot shows the loop length (in number of residues) on the y axis and receptor (sub)family types on the x axis.The length of the lines inside the violins is propor onal to the frac on of sequences in each (sub)family with the given loop lengths, with the 50% scale bars indicated at the top of each plot.Each plot shows five different violins: Gr43a subfamily (violet), Gr5a subfamily (green), Gr63a subfamily (red), all GRs (blue), and all ORs (orange).Dis nct le ers above each violin denote sta s cally dis nct classes, listed alphabe cally from highest median value to lowest such that adjacent categories bear adjacent le ers based on a Steel-Dwass test, p<0.01.(B) Top views of a single subunit from the agonist-free BmGr9 structure (le ) and the DmGr5a AlphaFold2 model (right) showing rela ve differences in length and structure of their S1-S2 and S3-S4 loops.