Why can we smell all kinds of smells? A very basic but extremely complex scientific question

The sense of smell is one of the earliest senses formed by the human body. Its importance may be overlooked because it is too common in our lives. The sense of smell is not only used when enjoying delicious food or sensing environmental dangers, but is also closely related to memory and emotions. So, why can we smell? This is a very basic, but extremely complex question. The exploration of olfactory receptors is the key to finding the answer.

Written by Chen Qingchao (Postdoctoral Fellow, MRC Laboratory of Molecular Biology, University of Cambridge)

In the diverse material world, there is a world that we cannot see or touch, but can feel. It may come from the fragrance of soil and grass after rain, or from the temptation of delicious food on the table. It even exists in memory, connecting the stream of emotions. This is the "world of smell".

There are millions of different smells, each made up of hundreds of chemical molecules with different properties. Why can we sense and distinguish such a complex variety of smells? This has long been one of the less explored but extremely important scientific questions in biology.

Figure 1. Odor molecules contained in the scent emitted by common fruits and vegetables (strawberries, tomatoes, and blueberries). Each circle and square represents an odor molecule. | Source: salk.edu

In fact, "feeling" and "discrimination" are two different biological problems: one is how our olfactory system perceives complex and diverse odor molecules; the other is how our nervous system decodes odor signals to form different olfactory perceptions. This article focuses on the first question and shares with you the exploration process of olfactory receptor structure research over the past few decades.

Searching for olfactory receptors

The sense of smell is one of the earliest senses to develop in the human body. It is a very complex sensory response. Through millions of olfactory nerves, we are able to perceive and distinguish a wide range of small molecular compounds with different structural properties, i.e. odor molecules, even at very low concentrations (micromolar or even nanomolar concentrations). [2]

The nasal mucosa of the human body is covered with a tissue called the olfactory epithelium, in which a large number of olfactory sensory neurons grow and are connected to each other. Olfactory sensory nerve cells extend to the mucus layer in the nasal cavity through cilia. The process of us smelling a certain smell is as follows (Figure 1): Odor molecules enter the nasal mucosa, are sensed by the primary cilia of the olfactory sensory neurons, thereby activating the olfactory nerve cells and generating chemical signals; these chemical signals trigger the nerve cells to generate electrical signals, which are then transmitted to the olfactory bulb through the olfactory nerves, and then to the olfactory cortex (the cortical area of ​​the brain responsible for olfactory processing). In the olfactory cortex, the brain analyzes and identifies the incoming olfactory information. Ultimately, the processing of olfactory nerve signals forms semantic representations that describe various odors, such as coffee, rose, mango, and so on.

Figure 2. Schematic diagram of the human olfactory system. From odor perception, signal transmission to final information processing. | Source: nobelprize.org

For a long time, a key question in the field of olfactory research is how cells sense complex and diverse odor molecules. A reasonable hypothesis is that there is a special protein on the olfactory sensory nerve cells, called "olfactory (odor) receptor" (Ordorant Receptor, OR), which is used to detect odor molecules. Scientists have been striving to find these special olfactory receptor proteins.

In the mid-1980s, a series of physiological and biochemical experiments conducted by different research groups showed that odor activation of olfactory sensory neurons is mediated by G protein-dependent pathways.

G proteins are a very important class of signal transduction molecules in cells. They work in conjunction with G protein-coupled receptors (GPCRs) to transmit signals generated by various signal factors such as hormones and neurotransmitters into cells, and further regulate the functions of enzymes, ion channels, transporters and various other proteins. In olfactory neurons, G proteins mediate the activation of adenylate cyclase, the increase in intracellular cyclic adenosine monophosphate (cAMP) concentration, the activation of cAMP-gated ion channels and neuronal depolarization [4].

During the same period, a number of olfactory-specific genes were cloned, including genes encoding G proteins and cAMP-gated ion channels, which further confirmed the important role of G protein signaling pathways in odor signal transduction. These studies strongly suggested that olfactory receptors are likely to be G protein-coupled receptors (GPCRs).

In 1991, Linda Buck and Richard Axel published a groundbreaking study in the journal Science, cloning and identifying the olfactory receptor GPCR gene family from rats for the first time [6]. Through further analysis, they also proved that these receptors are only expressed in rat olfactory epithelial cells, and not in eight other tissues (including the brain, retina, and liver). In addition, in order to estimate the size of the olfactory gene family, they further used a mixture of DNA as a probe to screen the rat genomic library. The screening results at the time showed that the rat haploid genome contained at least 500-1000 olfactory receptor genes.

Buck and Axel then worked independently to further discover the presence of olfactory receptor GPCR genes in human olfactory tissue and confirmed their important role in the human olfactory system.

These pioneering works laid an important foundation for our understanding and research of the mysterious sense of smell, for which the two won the 2004 Nobel Prize in Physiology or Medicine.

Figure 3. The 2004 Nobel Prize in Physiology or Medicine was jointly awarded to Richard Axel (left) and Linda B. Buck (right) for their "discoveries of odor receptors and the structure of the olfactory system." | Image source: nobelprize.org

After 2004, the completion of the Human Genome Project made it possible to identify and classify human olfactory receptor genes, further promoting the development of olfactory receptor research.

We now know that olfactory receptors are mainly G protein-coupled receptors (GPCRs) with seven transmembrane structures. GPCRs have more than 800 family members in the human body and are the largest family of cell surface receptors in eukaryotes. They are involved in the regulation of almost all life activities in the human body. For this reason, GPCRs have become "star molecules" in scientific research and important targets for drug development. Among all drugs approved by the U.S. Food and Drug Administration (FDA), about one-third work by targeting and regulating the activity of different GPCRs [7]. Among all GPCRs in the human body, about 400 members are classified as olfactory receptors, accounting for half of the GPCR members, making them the largest protein family.

The Dilemma of Olfactory Receptor Structural Elucidation

Since the first discovery of olfactory receptors in 1991, structural biologists have been committed to analyzing the structure of olfactory receptors to clarify their mechanism of recognizing odor molecules. However, in the past 30 years, the analysis of olfactory receptor structures has not progressed smoothly and has faced many challenges.

First, most human olfactory receptors are mainly expressed in nasal nerve cells, and the expression level is low. Therefore, it is difficult to obtain sufficient amount of protein (usually milligrams) directly from human tissue samples for structural analysis. The effect of heterologous expression (expression in animal cells or bacteria) is also not ideal. Not only is the expression level very low, but it also lacks biological activity due to misfolding.

Second, in order to resolve the protein structure of GPCR, we need to bind some specific high-affinity ligand molecules, that is, suitable odor molecules. However, due to the huge chemical diversity of odor molecules and the large number of olfactory receptors, there is currently a lack of an efficient method to determine which odor molecules a given olfactory receptor interacts with.

The academic community has gradually come to realize that each olfactory receptor can interact with a subset of all potential odor molecules, that one odor molecule can activate multiple olfactory receptors, and that different receptors have different affinities for different odor molecules. The complexity of this interaction has led to a large number of olfactory receptors not finding suitable odor molecule ligands, and these receptors are called "orphan receptors" [8]. Currently, a lot of "de-orphanization" research is underway to develop effective screening methods to find suitable ligands for orphan receptors. In addition, since most volatile odor molecules are hydrophobic molecules with low solubility, this greatly increases the difficulty of preparing odor molecule ligands.

Third, as an important molecule for signal perception and transduction on the cell membrane, GPCR is a highly dynamic protein molecule that constantly changes in various conformations such as inactive, semi-activated, activated, and coupled with different regulatory molecules. Therefore, like most other GPCRs, one of the difficulties in olfactory receptor purification is to stabilize the receptor protein in a specific conformation, which is very important for the formation of protein crystals.

In recent years, many research groups have developed many methods to stabilize different conformations of GPCRs, including but not limited to obtaining highly stable receptor mutants for protein crystallization through stability mutation; stabilizing the structure of GPCRs coupled to G proteins in a fully active state by combining "mini G proteins (miniGs)"; combining high-affinity small molecule ligands (including agonists, antagonists, inverse agonists, etc.); developing new nanobodies (Nanobodies) to stabilize different complex conformations of GPCRs, etc. For a specific GPCR, many different methods need to be tried to stabilize a specific conformation, which is a very time-consuming and labor-intensive process.

The dawn of hope: from insects to humans

Today, structural biology has entered the era of cryo-electron microscopy from crystal diffraction. In a complete single-particle cryo-electron microscopy technique, the purified protein is instantly frozen in a thin layer of non-crystalline vitreous ice, and then imaged by transmission electron microscopy to record data of hundreds of thousands to millions of protein particles - for three-dimensional reconstruction and precise modeling (Figure 4). Compared with traditional crystallographic methods, single-particle cryo-electron microscopy (Cryo-EM) has obvious advantages in resolving high-resolution structures of biological macromolecules, such as no need to obtain crystals, small sample amounts required, and a variety of sample preparation methods, etc., and has been widely used to resolve the complex structure of GPCRs and downstream proteins, which has brought hope to the resolution of olfactory receptor structures.

Figure 4. Single Particle Cryo-EM basic workflow: Place the purified protein sample on a grid and then vitrify it with liquid ethane. The protein particles embedded in the thin ice will have various random directions, which are imaged by transmission electron microscopy (TEM), and then three-dimensionally reconstructed through a series of image processing, ultimately obtaining a high-resolution protein cryo-EM structure. | Source: pdf.medrang.co.kr

In 2018, researchers from the Ruta Laboratory at Rockefeller University in the United States resolved the single-particle cryo-EM structure of Orco, an odor co-receptor of a parasitic wasp, at a resolution of nearly 3.5 Å [9]. Unlike mammals, insect odor receptors are not GPCRs, but gated ion channels, which are heteropolymeric ion channels composed of the odor receptor OR and the highly conserved co-receptor Orco. This ion channel is like a hole through which charged particles flow. It will only open when the receptor encounters its target odor molecule, thereby activating the olfactory sensory cells. For a long time, the scientific community has been controversial about whether Orco can function as an independent olfactory receptor, and no unified model of insect odor perception and signal transduction has been formed. This work shows for the first time the fine structure of the insect odor co-receptor Orco homotetramer, providing conclusive evidence to determine that "the insect olfactory co-receptor Orco can form a new type of heteropolymeric ligand-gated ion channel", obtaining structural analysis and confirming its function, providing important new insights into the understanding of insect peripheral olfactory mechanisms.

In 2021, another study from the Ruta lab resolved the cryo-EM structure of the olfactory receptor OR5 of a stonefly (Machilis hrabei) [10] (Figure 5). By comparing the structures of OR5 bound to three different odor molecules, the researchers found that the binding of odor molecules mainly relies on hydrophobic interactions and lacks the strict geometric constraints inherent in other intermolecular forces (such as hydrogen bonds) that often mediate ligand recognition.

Hydrophobic interaction is a force that stabilizes the three-dimensional structure of proteins, usually occurring between two or more non-polar amino acid residues. When they are in a polar environment (most commonly water), their "dislike" for water causes them to approach each other in a certain way so as to interact as little as possible with the polar environment. This non-specific weak interaction provides a new mechanism to explain "why one olfactory receptor can recognize different odorants", which is different from the classic "lock and key" model of many other receptor-ligand interactions. But the non-specificity of the OR5 receptor does not mean that it has no preference. Although it can bind to many different odor molecules, it is not sensitive to many other odor molecules. In addition, if a simple mutation is made to some amino acids in the binding pocket, that is, the receptor is re-changed, the receptor can bind to molecules that it originally disliked. This discovery also helps to explain why insects can evolve millions of odor receptors through mutation during evolution to adapt to the various living environments they encounter and form a unique lifestyle.

Figure 5. Cryo-EM structure of the olfactory receptor OR5 of the stonefeller (Machilis hrabei). When odor molecules bind to the olfactory receptor, the channel pore (blue) of the olfactory receptor expands (pink). | Image source: rockefeller.edu

The above structural biology studies on insect olfactory receptors have brought many new insights into our understanding of the mechanism of odor recognition. However, humans and insects are different after all. We urgently need high-resolution structures of human olfactory receptors to unveil the "veil" of human olfactory perception.

It was not until March 2023 that an article published in Nature magazine revealed for the first time the mystery of the human olfactory receptor structure[11].

In this work, the researchers chose an olfactory receptor called OR5E2. They chose this receptor because it is expressed not only in olfactory nerve cells, but also in other non-olfactory organs such as the prostate, indicating that it is easier to express in a heterologous system. In other words, it is easier to obtain sufficient protein.

The matching molecules of this receptor are also easy to obtain. Previous studies have shown that this receptor can bind and respond to the water-soluble short chain fatty acids (SCFAs) odor molecule - propionic acid. Short chain fatty acids are a type of signaling molecules produced by intestinal flora. They are volatile, have a special pungent smell, and play an important role in the occurrence and development of many diseases.

Additionally, OR5E2 is relatively conserved during evolution, possibly because they recognize odors that are critical to the survival of many species of animals, and the researchers infer that this olfactory receptor may be more constrained in evolution by stability.

In short, through these strategies, the researchers cleverly circumvented the challenges of low expression levels of most olfactory receptors, low solubility of most volatile odorants, and high instability of purified olfactory receptors. By fusion expression of mini G proteins, as well as combining Gβ1γ2 proteins and nanoantibodies Nb35, the researchers stabilized an activated state of OR5E2 bound to propionic acid and used cryo-electron microscopy to resolve its three-dimensional high-resolution structure (Figure 6).

Figure 6. 3D structure of human odorant receptor OR51E2 (green). The purple, red, and blue helices and tangles are G protein subunits that couple to the receptor, and the orange is the nanobody used to stabilize the structure. | Source: Kristina Armitage/Quanta Magazine; Sources: NIH/NIDCD; ArtBalitskiy/iStock; Alhontess/iStock

In this structure, the OR51E2 receptor locks the odor molecule propionic acid in a small closed binding pocket. In this small pocket, propionic acid binds to OR51E2 through two types of interactions: polar interactions (hydrogen bonds and ionic bonds), and nonspecific hydrophobic interactions. Therefore, the way OR51E2 binds odor molecules is different from that of insect odor-gated ion channels and seems to be more selective.

Many olfactory receptors are able to respond to a variety of chemically different odorants, but OR51E2 seems to bind only to short-chain fatty acids. So what factors determine this selectivity? Further analysis of this structure showed that OR51E2's selectivity for short-chain fatty acids stems from the volume of the closed binding pocket (31 Å), which can accommodate short-chain fatty acids such as acetate and propionate, but prevents longer fatty acid chains from binding. Therefore, the researchers believe that the volume of the binding pocket is an important selectivity factor for odor molecules.

As the first published activated structure of a human olfactory receptor bound to an odor molecule ligand, this is an exciting research result. It allows us to see for the first time how odor molecules bind to olfactory receptors, although it is not perfect in many aspects, such as the coupling between the receptor and G protein.

The binding of ligands to GPCRs usually causes conformational changes, which in turn couples the G proteins and further transmits signals to the G proteins. Under physiological conditions, mammalian olfactory receptors can bind to two highly homologous G proteins, Gαolf and Gαs. In this structure, the researchers did not couple Gαolf or Gαs, but instead used fusion expression of miniGαs, and combined Gβ1γ2 and nanoantibody Nb35 to stabilize the structure of the receptor and G protein heterotrimer. Although some interactions between olfactory receptors and G proteins have been found, this is not enough to explain the interaction mechanism with the real G proteins Gαolf and Gαs in vivo.

On May 24, 2023, Sun Jinpeng's laboratory at the School of Basic Medical Sciences of Shandong University published a work online in the journal Nature, systematically analyzing the structure of the mouse trace amine olfactory receptor TAAR9 (mTAAR9) recognizing four endogenous amine ligands (phenylethylamine, dimethylcyclohexylamine, cadaverine, spermidine) and coupling with downstream Gαs and Gαolf proteins [12].

Trace amine-associated receptors (TAARs) are a class of evolutionarily conserved G protein-coupled receptors in vertebrates that can sense trace amines at nanomolar concentrations. Trace amines are formed by decarboxylation of amino acids. For animals, they can serve as odor molecules that sense a series of stimuli, such as the presence of predators or prey, the approach of mating partners, and the spoilage of food, and can cause intra- or inter-species attraction or aversion reactions based on the odor. In recent years, more and more studies have shown that trace amines in the human body are associated with a variety of psychiatric disorders, and TAARs have therefore become potential new therapeutic targets for psychiatric diseases such as schizophrenia, depression, and drug addiction.

Figure 7. The structure of the mouse olfactory receptor mTAAR9 complex with different ligands and the trimer complex of Gas and Gaolf proteins. | Source: Nature

In this study, the researchers found that the olfactory receptor TAAR formed a pair of disulfide bonds between the N-terminus and the second extracellular segment, which has never been found in other GPCR receptors with known structures. Moreover, this pair of disulfide bonds is crucial for mTAAR9 to recognize ligands and stabilize the extracellular conformation of the receptor activation state.

A single TAAR olfactory receptor can recognize a variety of amine odor molecules, and the same amine odor molecule can also be recognized by multiple olfactory receptors. The complex nature of this interaction is an important basis for olfactory perception of amine molecules. This study discovered the universal structural motifs of mTAAR9 for recognizing amine odor molecules and the combined structural motifs for recognizing different amine odor molecules, providing new insights into the recognition of amine odor molecules.

It is worth noting that the researchers also resolved the molecular structure of the mTAAR9 receptor coupled to two downstream G proteins, Gαs and Gαolf. As the first experimentally determined complex structure of an olfactory receptor and Gαolf, this provides important insights into the full activation of mammalian olfactory receptors after downstream G protein coupling.

Future challenges

With the help of cryo-electron microscopy, the structural analysis of olfactory receptors has begun to show signs, and greater challenges are coming.

The above structure only reveals an activated conformation, but under physiological conditions, olfactory receptors are highly dynamic. With the rapid development of artificial intelligence in the field of protein structure prediction, researchers have also tried to show the dynamic changes of receptors through computer simulation to improve theoretical models, but this is not completely equivalent to the structural changes under real physiological conditions. We need to analyze more structures of olfactory receptors under different time dynamics, and develop high-resolution receptor protein dynamic monitoring methods to help us open the complete biological "black box" of olfactory perception.

In recent years, with the continuous development of sequencing technology, the expression of olfactory receptors has been found in more non-olfactory tissues, including the heart, respiratory tract, kidney, liver, lung, skin, brain and other parts. The expression of these olfactory receptors in non-olfactory tissues is both universal and specific. Studies have shown that olfactory receptors expressed outside the nasal cavity have specific biological functions in specific tissues [13]. Some studies have found that abnormal function of olfactory receptors is related to the occurrence and development of diseases such as nervous system diseases and tumors. Analyzing the physiological structure of these receptors in non-olfactory tissues provides new directions and challenges for the study of olfactory receptor structure. These olfactory receptors are also expected to become important drug targets in the future.

Back to the question at the beginning of this article: Why can our olfactory system sense and distinguish such complex and diverse smells? Scientifically, we still cannot fully answer this question, and as we study more and understand the structure of olfactory receptors more deeply, this question seems to become more complicated. How olfactory receptors selectively respond to odor molecules in the air is only part of the larger odor puzzle. Researchers still face a more complex challenge: understanding how the brain converts the electrochemical signals transmitted by the receptors into the perception of odor.

We still have a long way to go in understanding the mysteries of olfactory perception.

References

[1] https://pubmed.ncbi.nlm.nih.gov/28424010/

[2] https://academic.oup.com/nar/article/50/D1/D678/6362078

[3] https://www.ingentaconnect.com/content/ben/cn/2019/00000017/00000009/art00010

[4] https://www.ncbi.nlm.nih.gov/books/NBK55985/

[5] https://www.science.org/doi/10.1126/

[6] https://pubmed.ncbi.nlm.nih.gov/1840504/

[7] https://www.nature.com/articles/nrd.2017.178

[8] https://zh.wikipedia.org/wiki/%E5%AD%A4%E5%84%BF%E5%8F%97%E4%BD%93

[9] https://www.nature.com/articles/s41586-018-0420-8

[10] https://www.nature.com/articles/s41586-021-03794-8

[11] https://www.nature.com/articles/s41586-023-05798-y

[12] https://www.nature.com/articles/s41586-023-06106-4

[13] https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0055368

This article is supported by the Science Popularization China Starry Sky Project

Produced by: China Association for Science and Technology Department of Science Popularization

Producer: China Science and Technology Press Co., Ltd., Beijing Zhongke Xinghe Culture Media Co., Ltd.

Special Tips

1. Go to the "Featured Column" at the bottom of the menu of the "Fanpu" WeChat public account to read a series of popular science articles on different topics.

2. Fanpu provides a function to search articles by month. Follow the official account and reply with the four-digit year + month, such as "1903", to get the article index for March 2019, and so on.

Copyright statement: Personal forwarding is welcome. Any form of media or organization is not allowed to reprint or excerpt without authorization. For reprint authorization, please contact the backstage of the "Fanpu" WeChat public account.