Functional characterization of vertebrate odorant receptors (ORs), members of the G protein-coupled receptor (GPCR) family, is essential for understanding olfaction. However, the functional expression of ORs in heterologous cells is often challenging, at least partly caused by structural instability in non-olfactory cells. Antagonists have been shown to restore membrane expression of some non-olfactory GPCR mutants, likely by transient increase in structural stability upon antagonist binding. Based on this premise, we examined whether antagonists could enhance OR membrane expression in heterologous cells. Using phenyl salicylate (PES) on cells expressing the mouse OR Or11g7, we observed increased cell surface expression exceeding the effects of co-expression with the OR chaperone RTP1S. After removing the antagonist, Or11g7 retained normal agonist responsiveness. Similar enhancements in cell surface expression were observed for a human OR OR2T11 treated with its antagonists. These findings suggest that small-molecule antagonists act as pharmacological chaperones to stabilize OR conformation, enhancing surface expression in a manner similar to molecular chaperones. Our study reveals a novel role for odorant antagonists in OR biogenesis and may inform future research on olfactory training mechanisms.

How the olfactory system detects and distinguishes odorants with diverse physicochemical properties and molecular configurations remains poorly understood. Vertebrate animals perceive odours through G protein-coupled odorant receptors (ORs)1. In humans, around 400 ORs enable the sense of smell. The OR family comprises two main classes: class I ORs are tuned to carboxylic acids whereas class II ORs, which represent most of the human repertoire, respond to a wide variety of odorants2. A fundamental challenge in understanding olfaction is the inability to visualize odorant binding to ORs. Here we uncover molecular properties of odorant-OR interactions by using engineered ORs crafted using a consensus protein design strategy3. Because such consensus ORs (consORs) are derived from the 17 major subfamilies of human ORs, they provide a template for modelling individual native ORs with high sequence and structural homology. The biochemical tractability of consORs enabled the determination of four cryogenic electron microscopy structures of distinct consORs with specific ligand recognition properties. The structure of a class I consOR, consOR51, showed high structural similarity to the native human receptor OR51E2 and generated a homology model of a related member of the human OR51 family with high predictive power. Structures of three class II consORs revealed distinct modes of odorant-binding and activation mechanisms between class I and class II ORs. Thus, the structures of consORs lay the groundwork for understanding molecular recognition of odorants by the OR superfamily.

A central challenge in olfaction is understanding how the olfactory system detects and distinguishes odorants with diverse physicochemical properties and molecular configurations. Vertebrate animals perceive odors via G protein-coupled odorant receptors (ORs). In humans, ~400 ORs enable the sense of smell. The OR family is composed of two major classes: Class I ORs are tuned to carboxylic acids while Class II ORs, representing the vast majority of the human repertoire, respond to a wide variety of odorants. How ORs recognize chemically diverse odorants remains poorly understood. A fundamental bottleneck is the inability to visualize odorant binding to ORs. Here, we uncover fundamental molecular properties of odorant-OR interactions by employing engineered ORs crafted using a consensus protein design strategy. Because such consensus ORs (consORs) are derived from the 17 major subfamilies of human ORs, they provide a template for modeling individual native ORs with high sequence and structural homology. The biochemical tractability of consORs enabled four cryoEM structures of distinct consORs with unique ligand recognition properties. The structure of a Class I consOR, consOR51, showed high structural similarity to the native human receptor OR51E2 and yielded a homology model of a related member of the human OR51 family with high predictive power. Structures of three Class II consORs revealed distinct modes of odorant-binding and activation mechanisms between Class I and Class II ORs. Thus, the structures of consORs lay the groundwork for understanding molecular recognition of odorants by the OR superfamily.

Odorant receptor proteins (ORs) have highly variable cell surface expression levels. The majority of both human and murine ORs depend on chaperone proteins to traffic from the endoplasmic reticulum to the cell surface, while a limited subset of ORs express at high levels independently. Quantifying these heterogeneous expression levels is of high import for understanding the trafficking and stability of these integral-transmembrane proteins and for normalizing in vitro activation assays. Recognizable epitopes like the rhodopsin-tag can be inserted upstream of the N-termini in ORs to enable cell surface immunostaining and detection via flow cytometry. This method enables robust measurement and comparison of cell surface expression levels of different ORs. Our approach also facilitates the study of different chaperone proteins' effects on OR trafficking and expression.

Our sense of smell enables us to navigate a vast space of chemically diverse odour molecules. This task is accomplished by the combinatorial activation of approximately 400 odorant G protein-coupled receptors encoded in the human genome1-3. How odorants are recognized by odorant receptors remains unclear. Here we provide mechanistic insight into how an odorant binds to a human odorant receptor. Using cryo-electron microscopy, we determined the structure of the active human odorant receptor OR51E2 bound to the fatty acid propionate. Propionate is bound within an occluded pocket in OR51E2 and makes specific contacts critical to receptor activation. Mutation of the odorant-binding pocket in OR51E2 alters the recognition spectrum for fatty acids of varying chain length, suggesting that odorant selectivity is controlled by tight packing interactions between an odorant and an odorant receptor. Molecular dynamics simulations demonstrate that propionate-induced conformational changes in extracellular loop 3 activate OR51E2. Together, our studies provide a high-resolution view of chemical recognition of an odorant by a vertebrate odorant receptor, providing insight into how this large family of G protein-coupled receptors enables our olfactory sense.

Sex hormones alter the organization of the brain during early development and coordinate various behaviors throughout life. In zebra finches, song learning is limited to males, with the associated song learning brain pathways only maturing in males and atrophying in females. While this atrophy can be prevented by treating females with exogenous estrogen during early post-hatch development, the requirement of estrogen during normal male song system development is uncertain. For the first time in songbirds, we administered exemestane, a potent third generation estrogen synthesis inhibitor, from the day of hatching until adulthood in order to reassess the role of estrogen in song circuit development. We examined the behavior, brain anatomy, and transcriptomes of individual song nuclei in these pharmacologically manipulated animals. We found that males with long-term exemestane treatment had diminished male-specific plumage and impaired song learning, but minimal effect on song nuclei sizes and their specialized transcriptome. Consistent with prior findings, females with long-term estrogen treatment retained a functional song system with song nuclei that had specialized gene expression similar, but not identical to males. We also observed that different song nuclei responded to estrogen manipulation differently, with Area X in the striatum being the most altered by estrogen modulation. These findings support the hypothesis that song learning is an ancestral trait in both sexes that was subsequently suppressed in females of some species and that estrogen has come to play a critical role in modulating this suppression as well as refinement of song learning.