Ligand Recognition Research Articles

Multiple Myeloma Cells Shift the Fate of Cytolytic ILC2s Towards TIGIT-Mediated Cell Death

Background: Growing evidence attests to the multifaceted roles of group 2 innate lymphoid cells (ILC2s) in cancer immunity. They exhibit either pro- or anticancer activity depending on tumor type but their function in Multiple Myeloma (MM) is still not elucidated. Methods: The bone marrow (BM) and peripheral blood (PB) of patients (pts) with MM or precancerous conditions were collected, and specific properties of ILC2 subsets were assessed by flow cytometry. Results: By dissecting ILC2s according to c-Kit marker, we observed that NKp30 and NKG2D were mainly confined to c-Kithi ILC2s, while levels of DNAM-1 was significantly higher in fully mature c-Kitlo cells. Among the total MM-associated ILC2s (MM-ILC2s), we observed a significant increase in c-the Kitlo subset, but the expression of DNAM-1 in these cells was significantly reduced, especially in BM. Interestingly, MM-ILC2s from PB expressed granzyme B (GZMB), but its expression was impaired in BM-ILC2s. Accordingly, MM cells were susceptible to killing by MM-ILC2s derived from PB while eluding ILC2 surveillance in BM. Indeed, in MM-ILC2s derived from BM, the downregulation of DNAM-1 is accompanied by the upregulation of TIGIT, which mediate cell death in ILC2s upon recognition of the cognate ligands expressed by MM cells. These ILC2 changes appeared in clinical precursor conditions and eventually accumulated with disease progression. Conclusions: MM-ILC2s can act as cytolytic immune effectors that are fully competent in PB. However, MM cells shift ILC2 fate towards cell death in BM via the upregulation of TIGIT, thereby representing a potential therapeutic target to restore ILC2 antitumor activity.

Fluorescent Artificial Receptor for Dopamine based on Molecular Recognition-driven Dynamic Covalent Chemistry in a Lipid Nanoreactor.

Molecular recognition and detection of small bioactive molecules, like neurotransmitters, remain a challenge for chemists, whereas nature found an elegant solution in form of protein receptors. Here, we introduce a concept of a dynamic artificial receptor that synergically combines molecular recognition with dynamic imine bond formation inside a lipid nanoreactor, inducing a fluorescence response. The designed supramolecular system combines a lipophilic recognition ligand derived from a boronic acid, a fluorescent aldehyde based on push-pull styryl pyridine and a phenol-based catalyst. The recognition ligand specifically captures dopamine inside lipid nanodroplets and thus triggers imine bond formation with the aldehyde, producing the emission color change. The rational design of the fluorescent aldehyde, the catalyst and the recognition ligand allows dramatic acceleration of the imine bond formation required for rapid sensing of dopamine. The nanoprobe enables dopamine detection with micromolar sensitivity and singe-nanoprobe imaging of dopamine gradients through its robust two-color ratiometric response. It displays remarkable selectivity without interference of competing biogenic primary amines and biological media: blood serum, plasma, urine and cell lysate. The proposed concept of a dynamic artificial receptor offers a solution to the long-standing problem of molecular recognition and sensing of small molecules in complex biological media.

Fluorescent Artificial Receptor for Dopamine based on Molecular Recognition‐driven Dynamic Covalent Chemistry in a Lipid Nanoreactor

Molecular recognition and detection of small bioactive molecules, like neurotransmitters, remain a challenge for chemists, whereas nature found an elegant solution in form of protein receptors. Here, we introduce a concept of a dynamic artificial receptor that synergically combines molecular recognition with dynamic imine bond formation inside a lipid nanoreactor, inducing a fluorescence response. The designed supramolecular system combines a lipophilic recognition ligand derived from a boronic acid, a fluorescent aldehyde based on push‐pull styryl pyridine and a phenol‐based catalyst. The recognition ligand specifically captures dopamine inside lipid nanodroplets and thus triggers imine bond formation with the aldehyde, producing the emission color change. The rational design of the fluorescent aldehyde, the catalyst and the recognition ligand allows dramatic acceleration of the imine bond formation required for rapid sensing of dopamine. The nanoprobe enables dopamine detection with micromolar sensitivity and singe‐nanoprobe imaging of dopamine gradients through its robust two‐color ratiometric response. It displays remarkable selectivity without interference of competing biogenic primary amines and biological media: blood serum, plasma, urine and cell lysate. The proposed concept of a dynamic artificial receptor offers a solution to the long‐standing problem of molecular recognition and sensing of small molecules in complex biological media.

Structural insights into binding-site access and ligand recognition by human ABCB1

Abstract ABCB1 is a broad-spectrum efflux pump central to cellular drug handling and multidrug resistance in humans. However, how it is able to recognize and transport a wide range of diverse substrates remains poorly understood. Here we present cryo-EM structures of lipid-embedded human ABCB1 in conformationally distinct apo-, substrate-bound, inhibitor-bound, and nucleotide-trapped states at 3.4–3.9 Å resolution, in the absence of stabilizing antibodies or mutations. The substrate-binding site is located within one half of the molecule and, in the apo state, is obstructed by the transmembrane helix (TM) 4. Substrate and inhibitor binding are distinguished by major TM rearrangements and their ligand binding chemistry, with TM4 playing a central role in all conformational transitions. Furthermore, our data identify secondary structure-breaking residues that impart localized TM flexibility and asymmetry between the two transmembrane domains. The resulting structural changes and lipid interactions that are induced by substrate and inhibitor binding can predict substrate-binding profiles and may direct ABCB1 inhibitor design.

Decoding the Torsional Dynamics of Main-Chain Atoms Within CαNN Motif Facilitating Specific Anion Recognition.

The structural plasticity of proteins at the molecular level is largely dictated by backbone torsion angles, which play a critical role in ligand recognition and binding. To establish the anion-induced cooperative arrangement of the main-chain (mc) torsion, herein, we analyzed a set of naturally occurring CαNN motifs as "static models" for their anion-binding competence through docking and molecular dynamics simulations and decoded its torsion angle influenced mc-driven anion recognition potential. By comparing a pool of 20 distinct sets of CαNN motif with identical sequences in their "anion bound/present, aP" and "anion free/absent, aA" versions, we could discern that there exists a positive correlation between the "difference of anion residence time (ΔRT)" and "difference among the main-chain torsion angle" of the aP and aA population. Notably, the anion interaction with CαNNs is locally energetically favorable even in a context-free non-proteinaceous environment and if the difference of the mc-torsion angles involving the Cα-1, N0, N1 residues for a population is higher between the aP and aA state, the difference among the ligand RT is also greater. At the atomistic level, the accommodation of anion is highly synergistic and cooperatively sways the interacting mc-atom torsions. By comparing the clustering of H-bonding patterns, the free energy of binding, and RT in both states, we provide evidence that to establish favorable thermodynamics and kinetics of ligand accommodation in these short structural motifs, proper reorientation of local-mc governed by torsions is a prerequisite. Our findings position the CαNN motif as a promising scaffold for peptidomimetic design and emphasize the critical role of loop region dynamics in protein structure-function relationships.

Deciphering ligand and metal ion dependent intricate folding landscape of Vc2 c-di-GMP riboswitch aptamer.

Riboswitches are RNAs that recognize ligands and regulate gene expression. They are typically located in the untranslated region of bacterial messenger RNA and consist of an aptamer and an expression platform. In this study, we examine the folding pathway of the Vc2 (Vibrio cholerae) riboswitch aptamer domain, which targets the bacterial secondary messenger cyclic-di-GMP. We demonstrated by nuclear magnetic resonance (NMR)and isothermal titration calorimetry that the stable folding of the Vc2 riboswitch requires an adequate supply of Mg2+, Na+ and K+ ions. We found that Mg2+ has a crucial role in the pre-folding of the aptamer, while K+ is essential for establishing the long-range G-C interactions and stabilizing the ligand binding pocket. Precise imino proton assignments revealed the progressive folding of the aptamer. The results indicate that the P2 helix consists of weaker and more dynamic base pairs compared to the P1b helix, allowing the rearrangement of the base pairs in the P2 helix during the folding process required for effective ligand recognition. This study provides a profound understanding riboswitch architecture and dynamics at the atomic level under physiological conditions as well as structural information on apo-state RNA.

Recognition of Self and Viral Ligands by NK Cell Receptors

ABSTRACTNatural killer (NK) cells are essential elements of the innate immune response against tumors and viral infections. NK cell activation is governed by NK cell receptors that recognize both cellular (self) and viral (non‐self) ligands, including MHC, MHC‐related, and non‐MHC molecules. These diverse receptors belong to two distinct structural families, the C‐type lectin superfamily and the immunoglobulin superfamily. NK receptors include Ly49s, KIRs, LILRs, and NKG2A/CD94, which bind MHC class I (MHC‐I) molecules, and NKG2D, which binds MHC‐I paralogs such MICA and ULBP. Other NK receptors recognize tumor‐associated antigens (NKp30, NKp44, NKp46), cell–cell adhesion proteins (KLRG1, CD96), or genetically coupled C‐type lectin‐like ligands (NKp65, NKR‐P1). Additionally, cytomegaloviruses have evolved various immunoevasins, such as m157, m12, and UL18, which bind NK receptors and act as decoys to enable virus‐infected cells to escape NK cell‐mediated lysis. We review the remarkable progress made in the past 25 years in determining structures of representatives of most known NK receptors bound to MHC, MHC‐like, and non‐MHC ligands. Together, these structures reveal the multiplicity of solutions NK receptors have developed to recognize these molecules, and thereby mediate crucial interactions for regulating NK cytolytic activity by self and viral ligands.

Development of a His-Tag-mediated pull-down and quantification assay for G-quadruplex containing DNA sequences.

In this study, we developed a simple pull-down assay using peptide nucleic acids (PNAs) equipped with a His-Tag and a G-quadruplex (G4) ligand for the selective recognition and quantification of G4-forming DNA sequences. Efficient and specific target recovery was achieved using optimized buffer conditions and magnetic Ni-NTA beads, while quantification was realized by employing the enzyme-like properties of the G4/hemin complex. The assay was validated through HPLC analysis and adapted for a 96-well plate format. The results show that higher recovery can be achieved using His-Tag with Ni-NTA magnetic beads as compared to the more common biotin-streptavidin purification. The inclusion of the G4-ligand as an additional selectivity handle was shown to be beneficial for both recovery and selectivity.

Pretreatment-free aptasensing of lactoferrin in complex biological samples by portable electrophoretic mobility shift assay

Aptamers are attractive recognition ligands for sensing proteins due to their favorable affinity, specificity, stability, and easy synthesis. However, it is difficult to detect proteins directly in complex biological samples without sophisticated equipment or tedious sample pretreatment. Herein, we developed a portable electrophoretic mobility shift assay (EMSA) platform for direct protein aptasensing in complex biological samples. This portable EMSA consists of a mini agarose gel apparatus and a simple blue light transmission instrument integrated a smartphone camera. Lactoferrin (LF), which plays critical roles in food quality and dry eye diagnosis, was selected for feasibility verification. The successful direct determination of LF in dairy samples revealed that agarose gel not only provides separation power for the LF-aptamer complex but also functions as a physical barrier, excluding milk fat globules and casein micelles and further eliminating their interference. In addition, this pretreatment-free approach also exhibits good generality and multiplexity for other proteins assay by altering aptamer sequences. Therefore, the portable EMSA could be a reliable, affordable, user-friendly and pretreatment-free tool for protein aptasensing in biological and clinical samples for point-of-need diagnostics as well as food quality and safety.

Enhanced Molecular Imaging through a Versatile Peptide Nanofiber for Self-Assembly and Precise Recognition.

Designing molecules for multivalent targeting of specific disease markers can enhance binding stability which is critical in molecular imaging and targeted therapy. Through rational molecular design, the nanostructures formed by self-assembly of targeting peptides are expected to achieve multivalent targeting by increasing the density of recognition ligands. However, the balance between targeting peptide self-assembly and molecular recognition remains elusive. In this study, we designed a targeting-peptide-based imaging probe system TAP which consist of the signal unit, the recognition motif, the assembly motif and a Pro-leverage. It is verified that TAP could specifically binds to PD-L1-positive tumor cells in a multivalent manner to produce biological effects, and could also be combined with imaging probes through unique self-assembly strategies. By the balance between the peptide self-assembly and targeting recognition, the specificity and stability can be improved while the accumulation capacity of the probes at the tumor site can be greatly enhanced compared with the conventional strategy, thus reducing side effects, providing an effective tool for diagnostic and therapeutic integration of tumors.

The conformation of the nSrc specificity-determining loop in the Src SH3 domain is modulated by a WX conserved sequence motif found in SH3 domains.

Cellular signaling networks are modulated by multiple protein-protein interaction domains that coordinate extracellular inputs and processes to regulate cellular processes. Several of these domains recognize short linear motifs, or SLiMs, which are often highly conserved and are closely regulated. One such domain, the Src homology 3 (SH3) domain, typically recognizes proline-rich SLiMs and is one of the most abundant SLiM-binding domains in the human proteome. These domains are often described as quite versatile, and indeed, SH3 domains can bind ligands in opposite orientations dependent on target sequence. Furthermore, recent work has identified diverse modes of binding for SH3 domains and a wide variety of sequence motifs that are recognized by various domains. Specificity is often attributed to the RT and nSrc loops near the peptide-binding cleft in this domain family, particularly for Class I binding, which is defined as RT and nSrc loop interactions with the N-terminus of the ligand. Here, we used the Src and Abl SH3 domains as a model to further investigate the role of the RT and nSrc loops in SH3 specificity. We created chimeric domains with both the RT and nSrc loop sequences swapped between these SH3 domains, and used fluorescence anisotropy assays to test how relative binding affinities were affected for Src SH3- and Abl SH3-specific ligands. We also used Alphafold-Multimer to model our SH3:peptide complexes in combination with molecular dynamics simulations. We identified a position that contributes to the nSrc loop conformation in Src SH3, the amino acid immediately following a highly conserved Trp that creates a hydrophobic pocket critical for SH3 ligand recognition. We defined this as the WX motif, where X = Trp for Src and Cys for Abl. A broad importance of this position for modulating nSrc loop conformation in SH3 domains is suggested by analyses of previously deposited SH3 structures, multiple sequence alignment of SH3 domains in the human proteome, and our biochemical and computational data of mutant Src and Abl SH3 domains. Overall, our work uses experimental approaches and structural modeling to better understand specificity determinants in SH3 domains.

GPR180 is a new member of the Golgi-dynamics domain seven-transmembrane helix protein family

GOLD domain seven-transmembrane helix (GOST) proteins form a new protein family involved in trafficking of membrane-associated cargo. They share a characteristic extracellular/luminal Golgi-dynamics (GOLD) domain, possibly responsible for ligand recognition. Based on structural homology, GPR180 is a new member of this protein family, but little is known about the cellular role of GPR180. Here we show the X-ray structure of the N-terminal domain of GPR180 (1.9 Å) and can confirm the homology to GOLD domains. Using cellular imaging we show the localization of GPR180 in intracellular vesicular structures implying its exposure to acidic pH environments. With Hydrogen/Deuterium Exchange-Mass Spectrometry (HDX-MS) we identify pH-dependent conformational changes, which can be mapped to a putative ligand binding site in the transmembrane region. The results reveal GPR180’s role in intracellular vesicles and offer insights into the pH-dependent function of this conserved GOST protein.

Electroactive supramolecular nanoprobes for electrochemical detection of rituximab in non-Hodgkon’s lymphoma patients

Polymeric supramolecular assemblies (PSAs) exhibit excellent chemical stability, mechanical integrity, and multifunctionality, which could drive the development of biosensors. However, trapped by the significant differences in the physicochemical properties and structures of recognition ligands and signal tags, PSAs that simultaneously carry ligands and signals have rarely been reported for bioanalysis. In this study, an amphiphilic block copolymer (PVIM-b-PFMMA) was controllably synthesized by reversible addition fragmentation chain transfer (RAFT) radical polymerization for the first time. The resultant copolymer can self-assemble into highly electroactive supramolecular nanoprobes (eSNPs) with integrated target recognition and signal amplification functions. Compared to traditional electroactive probes based on conductive nanomaterials or enzyme catalysis, these versatile eSNPs avoid disadvantages including high cost, tedious modification, low load capacity and leakage of signal tags. For the rapid, sensitive and accurate monitoring of rituximab in biological fluids, a new electrochemical biosensor was constructed by coupling eSNPs with mimotope peptide CN14-modified screen-printed gold electrodes. The developed biosensor achieved a limit of detection (LOD) as low as 25 pg/mL and was successfully used to monitor rituximab concentration in a series of serum samples from non-Hodgkon’s lymphoma patients. Compared with commercial enzyme-linked immunosorbent assay (ELISA) and previously reported methods, the proposed biosensor demonstrated higher sensitivity, lower cost, simpler operation and more efficient preparation, owing to the unique superiority of eSNPs. Overall, this study presents a promising pathway for developing PSAs-based biosensors and their applications in bioanalysis.

Structure of scavenger receptor SCARF1 and its interaction with lipoproteins.

SCARF1 (scavenger receptor class F member 1, SREC-1 or SR-F1) is a type I transmembrane protein that recognizes multiple endogenous and exogenous ligands such as modified low-density lipoproteins (LDLs) and is important for maintaining homeostasis and immunity. But the structural information and the mechanisms of ligand recognition of SCARF1 are largely unavailable. Here, we solve the crystal structures of the N-terminal fragments of human SCARF1, which show that SCARF1 forms homodimers and its epidermal growth factor (EGF)-like domains adopt a long-curved conformation. Then, we examine the interactions of SCARF1 with lipoproteins and are able to identify a region on SCARF1 for recognizing modified LDLs. The mutagenesis data show that the positively charged residues in the region are crucial for the interaction of SCARF1 with modified LDLs, which is confirmed by making chimeric molecules of SCARF1 and SCARF2. In addition, teichoic acids, a cell wall polymer expressed on the surface of gram-positive bacteria, are able to inhibit the interactions of modified LDLs with SCARF1, suggesting the ligand binding sites of SCARF1 might be shared for some of its scavenging targets. Overall, these results provide mechanistic insights into SCARF1 and its interactions with the ligands, which are important for understanding its physiological roles in homeostasis and the related diseases.

Negative Charges, Not Necessary Phosphorylation, are Required for Ligand Recognition by 14-3-3 Proteins.

Protein-protein interactions involving 14-3-3 proteins regulate various cellular activities in normal and pathological conditions. These interactions have mostly been reported to be phosphorylation-dependent, but the 14-3-3 proteins also interact with unphosphorylated proteins. In this work, we investigated whether phosphorylation is required, or, alternatively, whether negative charges are sufficient for 14-3-3ϵ binding. We substituted the pThr residue of pT(502-510) peptide by residues with varying number of negative charges, and investigated binding of the peptides to 14-3-3ϵ using MD simulations and biophysical methods. We demonstrated that at least one negative charge is required for the peptides to bind 14-3-3ϵ while phosphorylation is not necessary, and that two negative charges are preferable for high affinity binding.

The chemoreceptor controlling the Wsp-like transduction pathway in Halomonas titanicae KHS3 binds and responds to purine derivatives.

The chemosensory pathway HtChe2 from the marine bacterium Halomonas titanicae KHS3 controls the activity of a diguanylate cyclase. Constitutive activation of this pathway results in colony morphology alterations and an increased ability to form biofilm. Such characteristics resemble the behavior of the Wsp pathway of Pseudomonas. In this work, we investigate the specificity of Htc10, the only chemoreceptor coded within the HtChe2 gene cluster. The purine derivatives guanine and hypoxanthine were identified as ligands of the recombinantly produced Htc10 ligand-binding domain, with dissociation constants in the micromolar range, and its structure was solved by X-ray protein crystallography. The sensor domain of Htc10 adopts a double Cache folding, with ligands bound to the membrane-distal pocket. A high-resolution structure of the occupied guanine-binding pocketallowed the identification of residues involved in ligand recognition. Such residues were validated by site-directed mutagenesis and isothermal titration calorimetry analyses of the protein variants. Moreover, heterologous expression of Htc10 in a Pseudomonas putida mutant lacking the native Wsp chemoreceptor promoted biofilm formation, a phenotype that was further enhanced by Htc10-specific ligands. To our knowledge, this is the first description of binding specificity of a chemoreceptor that controls the activity of an associated diguanylate cyclase, opening the way for dynamic studies of the signaling behavior of this kind of sensory complex.

Stable and Minimum Size Solubilization of Membrane Proteins with Cocktails of Phospholipid Analogues.

Membrane proteins (MPs) play important roles in various cellular processes and are major targets for drugs. Solubilization of MPs is often needed for structural and biophysical studies. For high-resolution nuclear magnetic resonance measurements, there is a size limit of the sample (<100 kDa), and a high thermal stability at an increased temperature is required. Furthermore, lipid bilayer-like environments are desirable to preserve the native states of MPs. However, existing solubilization techniques do not fulfill these requirements at the same time. In this study, we combined two phospholipid analogues as a solubilizer and stabilizer to isolate MPs. This method maintained bacteriorhodopsin (bR) extracted from purple membranes in its native state for 7 d at 40 °C. The solubility was comparable to that of conventional detergents for MPs, and the thermal stability of the solubilizate was the best among them. The increase in the molecular size caused by the solubilization of bR was only 20 kDa, indicating that 20 phospholipid analogue molecules were sufficient to solubilize one bR molecule. 15N-1H heteronuclear single quantum coherence spectra of solubilized 2H- and 15N-labeled bR gave ∼80% of the expected peaks. In addition, the lysate of human neuropeptide Y2 receptor-expressing mammalian cells exhibited ligand recognition for 7 d at 37 °C, suggesting that this technique can be used for ligand screening. Moreover, the structure of the single membrane-spanning M2 protein of the influenza A virus expressed in Escherichia coli was stably maintained for 7 d at 40 °C. Thus, our method is promising for various MP studies.

Structural mechanisms of potent lysophosphatidic acid receptor 1 activation by nonlipid basic agonists.

Lysophosphatidic acid receptor 1 (LPA1) is one of the G protein-coupled receptors activated by the lipid mediator, lysophosphatidic acid (LPA). LPA1 is associated with a variety of diseases, and LPA1 agonists have potential therapeutic value for treating obesity and depression. Although potent nonlipid LPA1 agonists have recently been identified, the mechanisms of nonlipid molecule-mediated LPA1 activation remain unclear. Here, we report a cryo-electron microscopy structure of the human LPA1-Gi complex bound to a nonlipid basic agonist, CpY, which has 30-fold higher agonistic activity as compared with LPA. Structural comparisons of LPA1 with other lipid GPCRs revealed that the negative charge in the characteristic binding pocket of LPA1 allows the selective recognition of CpY, which lacks a polar head. In addition, our structure show that the ethyl group of CpY directly pushes W2716.48 to fix the active conformation. Endogenous LPA lacks these chemical features, which thus represent the crucial elements of nonlipid agonists that potently activate LPA1. This study provides detailed mechanistic insights into the ligand recognition and activation of LPA1 by nonlipid agonists, expanding the scope for drug development targeting the LPA receptors.

The structural basis for high-affinity c-di-GMP binding to the GSPII-B domain of the traffic ATPase PilF from Thermus thermophilus

c-di-GMP is an important second messenger in bacteria regulating e. g. motility, biofilm formation, cell wall biosynthesis, infectivity and natural transformability. It binds to a multitude of intracellular receptors. This includes proteins containing general secretory pathway II (GSPII) domains such as the N-terminal domain of the Vibrio cholerae ATPase MshE (MshEN) which binds c-di-GMP with two copies of a 24-amino acids sequence motif. The traffic ATPase PilF from Thermus thermophilus is important for type IV pilus biogenesis, twitching motility, surface attachment and natural DNA-uptake and contains three consecutive homologous GPSII domains.We show that only two of these domains bind c-di-GMP and define the structural basis for the exceptional high affinity of the GSPII-B domain for c-di-GMP, which is 83fold higher than that of the prototypical MshEN domain. Our work establishes an extended consensus sequence for the c-di-GMP binding motif and highlights the role of hydrophobic residues for high-affinity recognition of c-di-GMP. Our structure is the first example for a c-di-GMP-binding domain not relying on arginine residues for ligand recognition. We also show that c-di-GMP binding induces local unwinding of an α-helical turn as well as subdomain reorientation to reinforce intermolecular contacts between c-di-GMP and the C-terminal subdomain. Abolishing c-di-GMP-binding to GSPII-B reduces twitching motility and surface attachment but not natural DNA-uptake.Overall, our work contributes to a better characterization of c-di-GMP binding in this class of effector domains, allows the prediction of high-affinity c-di-GMP-binding family members and advances our understanding of the importance of c-di-GMP binding for T4P-related functions.

Engineered odorant receptors illuminate the basis of odour discrimination.

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.