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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 - Nat Commun (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA [2] Department of Informatics, Technische Universität München, 85748 Garching, Germany.

ABSTRACT
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.

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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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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).

Mentions: To validate the predictive power of our ECs in identifying important residues in insect ORs, we focussed on the N-terminal cytoplasmic region of ORCO, whose role (if any) is unknown. We generated a version of this receptor (ORCO6Mut) bearing mutations in six of the top-ranked constrained residues in the sequence of D. melanogaster ORCO (A23, M24, F30, M31, H32, N33) (Supplementary Data 10); several of these residues feature as pairs in the top ranked ORCO ECs (i.e. M24-F30, M24-M31, M24-H32; Supplementary Data 6). We also generated an ORCO mutant bearing a small deletion spanning these residues (ORCOΔ23-33). Wildtype and mutant ORCO proteins were expressed in Xenopus oocytes for functional analysis by two-electrode voltage clamp recording of ligand-evoked current responses20. We first co-expressed ORCO with OR47a or OR85b, whose most potent agonists are pentyl acetate and 2-heptanone, respectively18, 30, 36. Wild type ORCO co-expressed with these receptors can reconstitute dose-dependent odour-evoked currents (Fig. 4a-b). By contrast, ORCO6Mut and ORCOΔ23-33 have highly diminished or abolished response to these odours, respectively (Fig. 4a-b). In a second set of experiments, we tested current responses of wildtype and mutant ORCOs to VUAA1, an artificial small-molecule agonist that can activate this co-receptor when expressed alone (without a tuning OR partner)37. Wildtype ORCO produced robust currents upon VUAA1 presentation, but neither mutant protein responded to this ligand (Fig. 4c). Taken together, our computational and experimental data support a previously unappreciated function for the ORCO N-terminus; that the tuning receptor OR67d is also sensitive to a mutation in this region33, suggests that this sequence may have a conserved role across the OR family. Determining the precise contribution of the N-terminus to receptor function – for example, in folding, trafficking and/or complex assembly – will nevertheless require further experimentation beyond the scope of this study.


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 - Nat Commun (2015)

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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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).
Mentions: To validate the predictive power of our ECs in identifying important residues in insect ORs, we focussed on the N-terminal cytoplasmic region of ORCO, whose role (if any) is unknown. We generated a version of this receptor (ORCO6Mut) bearing mutations in six of the top-ranked constrained residues in the sequence of D. melanogaster ORCO (A23, M24, F30, M31, H32, N33) (Supplementary Data 10); several of these residues feature as pairs in the top ranked ORCO ECs (i.e. M24-F30, M24-M31, M24-H32; Supplementary Data 6). We also generated an ORCO mutant bearing a small deletion spanning these residues (ORCOΔ23-33). Wildtype and mutant ORCO proteins were expressed in Xenopus oocytes for functional analysis by two-electrode voltage clamp recording of ligand-evoked current responses20. We first co-expressed ORCO with OR47a or OR85b, whose most potent agonists are pentyl acetate and 2-heptanone, respectively18, 30, 36. Wild type ORCO co-expressed with these receptors can reconstitute dose-dependent odour-evoked currents (Fig. 4a-b). By contrast, ORCO6Mut and ORCOΔ23-33 have highly diminished or abolished response to these odours, respectively (Fig. 4a-b). In a second set of experiments, we tested current responses of wildtype and mutant ORCOs to VUAA1, an artificial small-molecule agonist that can activate this co-receptor when expressed alone (without a tuning OR partner)37. Wildtype ORCO produced robust currents upon VUAA1 presentation, but neither mutant protein responded to this ligand (Fig. 4c). Taken together, our computational and experimental data support a previously unappreciated function for the ORCO N-terminus; that the tuning receptor OR67d is also sensitive to a mutation in this region33, suggests that this sequence may have a conserved role across the OR family. Determining the precise contribution of the N-terminus to receptor function – for example, in folding, trafficking and/or complex assembly – will nevertheless require further experimentation beyond the scope of this study.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA [2] Department of Informatics, Technische Universität München, 85748 Garching, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus