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Nat Commun
2015 Jan 13;6:6077. doi: 10.1038/ncomms7077.
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Amino acid coevolution reveals three-dimensional structure and functional domains of insect odorant receptors.
Hopf TA
,
Morinaga S
,
Ihara S
,
Touhara K
,
Marks DS
,
Benton R
.
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Insect odorant receptors (ORs) comprise an enormous protein family that translates environmental chemical signals into neuronal electrical activity. These heptahelical receptors are proposed to function as ligand-gated ion channels and/or to act metabotropically as G protein-coupled receptors (GPCRs). Resolving their signalling mechanism has been hampered by the lack of tertiary structural information and primary sequence similarity to other proteins. We use amino acid evolutionary covariation across these ORs to define restraints on structural proximity of residue pairs, which permit de novo generation of three-dimensional models. The validity of our analysis is supported by the location of functionally important residues in highly constrained regions of the protein. Importantly, insect OR models exhibit a distinct transmembrane domain packing arrangement to that of canonical GPCRs, establishing the structural unrelatedness of these receptor families. The evolutionary couplings and models predict odour binding and ion conduction domains, and provide a template for rationale structure-activity dissection.
205202 European Research Council, 615094 European Research Council, R01 GM106303 NIGMS NIH HHS , ERC_205202 European Research Council, ERC_615094 European Research Council
Figure 2. An Evolutionary Coupling-based insect OR model has a heptahelical packing arrangement distinct from G-protein coupled receptorsTop: Comparison of the helical packing arrangements of insect ORs, a G protein-coupled receptor (GPCR) and the Adiponectin receptor 1 (AR1); the structures are colour-coded from N-terminus (blue) to C-terminus (red). The OR is the top-ranked model of OR85b (see Fig. 1c); the GPCR is the crystal structure of the β2-adrenergic receptor (PDB 2RH155); the AR1 structure is the top-ranked model described previously28. As the N-termini of ORs and AR1s are located in the cytosol, but the N-terminus of GPCRs is located extracellularly (or lumenally), the GPCR structure is shown in the opposite orientation so that the packing arrangements are visually comparable. Structures were rotated so that the positioning of TMH1 and TMH2 agrees, as far as possible, between the different molecules. Bottom: corresponding simplified two-dimensional representations of the helical contacts highlighting the difference between the helical packing arrangement of insect ORs when compared to GPCRs and AR1s.
Figure 3. Highly coupled OR residues coincide with experimentally characterised functional regions(a) Positions with above-average EC strength (top 25% of sites, blue spheres) on the top-ranked model of OR85b (140_12; Supplementary Data 13) (membrane-integral side view). Strongly coupled residues cluster around three regions of the model: (i) N-terminus, (ii) extracellular loop 2, (iii) intracellular loop 3 and along the span of TMH7. Many of the strongly-coupled TMH7 residues do not feature in the contact map (Fig. 1b), because they were excluded as structural contacts with our standard transmembrane clash filter.(b) Experimentally characterised residues in different OR and ORCO proteins (spheres coloured by different shades of blue according to functional categorisation; Supplementary Table 2) mapped onto the 3D model of OR85b (140_12), based on a sequence alignment of OR and ORCO sequences (Supplementary Data 2). Amongst many residues whose mutation have general deleterious effects on ion channel function, only a few residues influence ion selectivity (which are strong candidates for pore-lining residues), including two sites in ORCO (double asterisks marking residues at the extracellular end of TMH6 and TMH7) and two in a tuning OR (B. mori OR1) (single asterisks marking residues at the intracellular end of TMH5 and TMH6) (see text and Supplemental Table 2 for details).
Figure 4. Functional analysis of the ORCO N-terminus(a-c) Left: Representative whole cell current traces to the indicated stimuli with two-electrode voltage-clamp in Xenopus oocytes injected with cRNAs for the indicated combinations of wildtype or mutant D. melanogaster ORs. ORCO6Mut contains amino acid substitutions in the six top-ranked N-terminal residues (A23S, M24A, F30A, M31A, H32A, N33A); ORCOΔ23-33 bears a deletion of this region (A23-N33). Ligand solutions were applied for 3 s (arrowheads). Right: Quantification of current amplitudes (mean ± SEM; n = 5 oocytes for wildtype ORCO and ORCO6Mut; n = 4 for ORCOΔ23-33).
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