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Protease-activated receptors (PARs) are a unique group within the G protein-coupled receptor superfamily, orchestrating cellular responses to extracellular proteases via enzymatic cleavage, which triggers intracellular signaling pathways. Protease-activated receptor 1 (PAR1) is a key member of this family and is recognized as a critical pharmacological target for managing thrombotic disorders. In this study, we present cryo-electron microscopy structures of PAR1 in its activated state, induced by its natural tethered agonist (TA), in complex with two distinct downstream proteins, the Gqand Giheterotrimers, respectively. The TA peptide is positioned within a surface pocket, prompting PAR1 activation through notable conformational shifts. Contrary to the typical receptor activation that involves the outward movement of transmembrane helix 6 (TM6), PAR1 activation is characterized by the simultaneous downward shift of TM6 and TM7, coupled with the rotation of a group of aromatic residues. This results in the displacement of an intracellular anion, creating space for downstream G protein binding. Our findings delineate the TA recognition pattern and highlight a distinct role of the second extracellular loop in forming β-sheets with TA within the PAR family, a feature not observed in other TA-activated receptors. Moreover, the nuanced differences in the interactions between intracellular loops 2/3 and the Gα subunit of different G proteins are crucial for determining the specificity of G protein coupling. These insights contribute to our understanding of the ligand binding and activation mechanisms of PARs, illuminating the basis for PAR1’s versatility in G protein coupling.

The noradrenaline transporter has a pivotal role in regulating neurotransmitter balance and is crucial for normal physiology and neurobiology1. Dysfunction of noradrenaline transporter has been implicated in numerous neuropsychiatric diseases, including depression and attention deficit hyperactivity disorder2. Here we report cryo-electron microscopy structures of noradrenaline transporter in apo and substrate-bound forms, and as complexes with six antidepressants. The structures reveal a noradrenaline transporter dimer interface that is mediated predominantly by cholesterol and lipid molecules. The substrate noradrenaline binds deep in the central binding pocket, and its amine group interacts with a conserved aspartate residue. Our structures also provide insight into antidepressant recognition and monoamine transporter selectivity. Together, these findings advance our understanding of noradrenaline transporter regulation and inhibition, and provide templates for designing improved antidepressants to treat neuropsychiatric disorders.

Therapeutic antibodies are at the forefront of biotherapeutics, valued for their high target specificity and binding affinity. Despite their potential, optimizing antibodies for superior efficacy presents significant challenges in both monetary and time costs. Recent strides in computational and artificial intelligence (AI), especially generative diffusion models, have begun to address these challenges, offering novel approaches for antibody design. This review delves into specific diffusion-based generative methodologies tailored for antibody design tasks, de novo antibody design, and optimization of complementarity-determining region (CDR) loops, along with their evaluation metrics. We aim to provide an exhaustive overview of this burgeoning field, making it an essential resource for leveraging diffusion-based generative models in antibody design endeavors.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors, with many GPCRs having crucial roles in endocrinology and metabolism. Cryogenic electron microscopy (cryo-EM) has revolutionized the field of structural biology, particularly regarding GPCRs, over the past decade. Since the first pair of GPCR structures resolved by cryo-EM were published in 2017, the number of GPCR structures resolved by cryo-EM has surpassed the number resolved by X-ray crystallography by 30%, reaching >650, and the number has doubled every ~0.63 years for the past 6 years. At this pace, it is predicted that the structure of 90% of all human GPCRs will be completed within the next 5–7 years. This Review highlights the general structural features and principles that guide GPCR ligand recognition, receptor activation, G protein coupling, arrestin recruitment and regulation by GPCR kinases. The Review also highlights the diversity of GPCR allosteric binding sites and how allosteric ligands could dictate biased signalling that is selective for a G protein pathway or an arrestin pathway. Finally, the authors use the examples of glycoprotein hormone receptors and glucagon-like peptide 1 receptor to illustrate the effect of cryo-EM on understanding GPCR biology in endocrinology and metabolism, as well as on GPCR-related endocrine diseases and drug discovery.

The AT-rich interaction domain (ARID) family of DNA-binding proteins is a group of transcription factors and chromatin regulators with a highly conserved ARID domain that recognizes specific AT-rich DNA sequences. Dysfunction of ARID family members has been implicated in various human diseases including cancers and intellectual disability. Among them, ARID3a has gained increasing attention due to its potential involvement in autoimmunity. In this article we provide an overview of the ARID family, focusing on the structure and biological functions of ARID3a. It explores the role of ARID3a in autoreactive B cells and its contribution to autoimmune diseases such as systemic lupus erythematosus and primary biliary cholangitis. Furthermore, we also discuss the potential for drug discovery targeting ARID3a and present a plan for future research in this field.

Phosphorylation of G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) desensitizes G-protein signalling and promotes arrestin signalling, which is also modulated by biased ligands1–6. The molecular assembly of GRKs on GPCRs and the basis of GRK-mediated biased signalling remain largely unknown owing to the weak GPCR–GRK interactions. Here we report the complex structure of neurotensin receptor 1 (NTSR1) bound to GRK2, Gαqand the arrestin-biased ligand SBI-5537. The density map reveals the arrangement of the intact GRK2 with the receptor, with the N-terminal helix of GRK2 docking into the open cytoplasmic pocket formed by the outward movement of the receptor transmembrane helix 6, analogous to the binding of the G protein to the receptor. SBI-553 binds at the interface between GRK2 and NTSR1 to enhance GRK2 binding. The binding mode of SBI-553 is compatible with arrestin binding but clashes with the binding of Gαqprotein, thus providing a mechanism for its arrestin-biased signalling capability. In sum, our structure provides a rational model for understanding the details of GPCR–GRK interactions and GRK2-mediated biased signalling.

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) are two endogenous hormones recognized by PTH receptor-1 (PTH1R), a member of class B G protein- coupled receptors (GPCRs). Both PTH and PTHrP analogs including teriparatide and abaloparatide are approved drugs for osteoporosis, but they exhibit distinct pharmacology. Here we report two cryo-EM structures of human PTH1R bound to PTH and PTHrP in the G protein-bound state at resolutions of 2.62 Å and 3.25 Å, respectively. Detailed analysis of these structures uncovers both common and unique features for the agonism of PTH and PTHrP. Molecular dynamics (MD) simulation together with site-directed mutagenesis studies reveal the molecular basis of endogenous hormones recognition specificity and selectivity to PTH1R. These results provide a rational template for the clinical use of PTH and PTHrP analogs as an anabolic therapy for osteoporosis and other disorders.

Class B G-protein-coupled receptors (GPCRs), including glucagon-like peptide 1 receptor (GLP1R) and parathyroid hormone 1 receptor (PTH1R), are important drug targets1–5. Injectable peptide drugs targeting these receptors have been developed, but orally available small-molecule drugs remain under development6,7. Here we report the high-resolution structure of human PTH1R in complex with the stimulatory G protein (Gs) and a small-molecule agonist, PCO371, which reveals an unexpected binding mode of PCO371 at the cytoplasmic interface of PTH1R with Gs. The PCO371-binding site is totally different from all binding sites previously reported for small molecules or peptide ligands in GPCRs. The residues that make up the PCO371-binding pocket are conserved in class B GPCRs, and a single alteration in PTH2R and two residue alterations in GLP1R convert these receptors to respond to PCO371. Functional assays reveal that PCO371 is a G-protein-biased agonist that is defective in promoting PTH1R-mediated arrestin signalling. Together, these results uncover a distinct binding site for designing small-molecule agonists for PTH1R and possibly other members of the class B GPCRs and define a receptor conformation that is specific only for G-protein activation but not arrestin signalling. These insights should facilitate the design of distinct types of class B GPCR small-molecule agonist for various therapeutic indications.

The dopaminergic system, including five dopamine receptors (D1R to D5R), plays essential roles in the central nervous system (CNS); and ligands that activate dopamine receptors have been used to treat many neuropsychiatric disorders, including Parkinson’s Disease (PD) and schizophrenia. Here, we report cryo-EM structures of all five subtypes of human dopamine receptors in complex with G protein and bound to the pan-agonist, rotigotine, which is used to treat PD and restless legs syndrome. The structures reveal the basis of rotigotine recognition in different dopamine receptors. Structural analysis together with functional assays illuminate determinants of ligand polypharmacology and selectivity. The structures also uncover the mechanisms of dopamine receptor activation, unique structural features among the five receptor subtypes, and the basis of G protein coupling specificity. Our work provides a comprehensive set of structural templates for the rational design of specific ligands to treat CNS diseases targeting the dopaminergic system.

The complement receptors C3aR and C5aR1, whose signaling is selectively activated by anaphylatoxins C3a and C5a, are important regulators of both innate and adaptive immune responses. Dysregulations of C3aR and C5aR1 signaling lead to multiple inflammatory disorders, including sepsis, asthma and acute respiratory distress syndrome. The mechanism underlying endogenous anaphylatoxin recognition and activation of C3aR and C5aR1 remains elusive. Here we reported the structures of C3a-bound C3aR and C5a-bound C5aR1 as well as an apo-C3aR structure. These structures, combined with mutagenesis analysis, reveal a conserved recognition pattern of anaphylatoxins to the complement receptors that is different from chemokine receptors, unique pocket topologies of C3aR and C5aR1 that mediate ligand selectivity, and a common mechanism of receptor activation. These results provide crucial insights into the molecular understanding of C3aR and C5aR1 signaling and structural templates for rational drug design for treating inflammation disorders.

Trace amine-associated receptor 1 (TAAR1), the founding member of a nine-member family of trace amine receptors, is responsible for recognizing a range of biogenic amines in the brain, including the endogenous β-phenylethylamine (β-PEA)1as well as methamphetamine2, an abused substance that has posed a severe threat to human health and society3. Given its unique physiological role in the brain, TAAR1 is also an emerging target for a range of neurological disorders including schizophrenia, depression and drug addiction2,4,5. Here we report structures of human TAAR1–G-protein complexes bound to methamphetamine and β-PEA as well as complexes bound to RO5256390, a TAAR1-selective agonist, and SEP-363856, a clinical-stage dual agonist for TAAR1 and serotonin receptor 5-HT1AR (refs. 6,7). Together with systematic mutagenesis and functional studies, the structures reveal the molecular basis of methamphetamine recognition and underlying mechanisms of ligand selectivity and polypharmacology between TAAR1 and other monoamine receptors. We identify a lid-like extracellular loop 2 helix/loop structure and a hydrogen-bonding network in the ligand-binding pockets, which may contribute to the ligand recognition in TAAR1. These findings shed light on the ligand recognition mode and activation mechanism for TAAR1 and should guide the development of next-generation therapeutics for drug addiction and various neurological disorders.

Autoimmune diseases (AIDs) arise from a breakdown in immunological self-tolerance, wherein the adaptive immune system mistakenly attacks healthy cells, tissues and organs. AIDs impose excessive treatment costs and currently rely on non-specific and universal immunosuppression, which only offer symptomatic relief without addressing the underlying causes. AIDs are driven by autoantigens, targeting the autoantigens holds great promise in transforming the treatment of these diseases. To achieve this goal, a comprehensive understanding of the pathogenic mechanisms underlying different AIDs and the identification of specific autoantigens are critical. In this review, we categorize AIDs based on their underlying causes and compile information on autoantigens implicated in each disease, providing a roadmap for the development of novel immunotherapy regimens. We will focus on type 1 diabetes (T1D), which is an autoimmune disease characterized by irreversible destruction of insulin-producing β cells in the Langerhans islets of the pancreas. We will discuss insulin as possible autoantigen of T1D and its role in T1D pathogenesis. Finally, we will review current treatments of TID and propose a potentially effective immunotherapy targeting autoantigens.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has brought an unprecedented public health crisis and persistently threatens to humanity. With tireless efforts from scientists around the world, understanding of the biology of coronavirus has been greatly enhanced over the past 2 years. Structural biology has demonstrated its powerful impact on uncovering structures and functions for the vast majority of SARS-CoV-2 proteins and guided the development of drugs and vaccines against COVID-19. In this review, we summarize current progress in the structural biology of SARS-CoV-2 and discuss important biological issues that remain to be addressed. We present the examples of structure-based design of Pfizer’s novel anti-SARS-CoV-2 drug PF-07321332 (Paxlovid), Merck’s nucleotide inhibitor molnupiravir (Lagevrio), and VV116, an oral drug candidate for COVID-19. These examples highlight the importance of structure in drug discovery to combat COVID-19. We also discussed the recent variants of Omicron and its implication in immunity escape from existing vaccines and antibody therapies.

Thyroid-stimulating hormone (TSH), through activation of its G-protein-coupled thyrotropin receptor (TSHR), controls the synthesis of thyroid hormone—an essential metabolic hormone1–3. Aberrant signalling of TSHR by autoantibodies causes Graves’ disease (hyperthyroidism) and hypothyroidism, both of which affect millions of patients worldwide4. Here we report the active structures of TSHR with TSH and the activating autoantibody M225, both bound to the allosteric agonist ML-1096, as well as an inactivated TSHR structure with the inhibitory antibody K1-707. Both TSH and M22 push the extracellular domain (ECD) of TSHR into an upright active conformation. By contrast, K1-70 blocks TSH binding and cannot push the ECD into the upright conformation. Comparisons of the active and inactivated structures of TSHR with those of the luteinizing hormone/choriogonadotropin receptor (LHCGR) reveal a universal activation mechanism of glycoprotein hormone receptors, in which a conserved ten-residue fragment (P10) from the hinge C-terminal loop mediates ECD interactions with the TSHR transmembrane domain8. One notable feature is that there are more than 15 cholesterols surrounding TSHR, supporting its preferential location in lipid rafts9. These structures also highlight a similar ECD-push mechanism for TSH and autoantibody M22 to activate TSHR, therefore providing the molecular basis for Graves’ disease.

The Omicron BA.2 variant has become a dominant infective strain worldwide. Receptor binding studies show that the Omicron BA.2 spike trimer exhibits 11-fold and 2-fold higher potency in binding to human ACE2 than the spike trimer from the wildtype (WT) and Omicron BA.1 strains. The structure of the BA.2 spike trimer complexed with human ACE2 reveals that all three receptor-binding domains (RBDs) in the spike trimer are in open conformation, ready for ACE2 binding, thus providing a basis for the increased infectivity of the BA.2 strain. JMB2002, a therapeutic antibody that was shown to efficiently inhibit Omicron BA.1, also shows potent neutralization activities against Omicron BA.2. In addition, both BA.1 and BA.2 spike trimers are able to bind to mouse ACE2 with high potency. In contrast, the WT spike trimer binds well to cat ACE2 but not to mouse ACE2. The structures of both BA.1 and BA.2 spike trimer bound to mouse ACE2 reveal the basis for their high affinity interactions. Together, these results suggest a possible evolution pathway for Omicron BA.1 and BA.2 variants via a human-cat-mouse-human circle, which could have important implications in establishing an effective strategy for combating SARS-CoV-2 viral infections.

Bombesin receptor subtype-3 (BRS3) is an important orphan G protein-coupled receptor that regulates energy homeostasis and insulin secretion. As a member of the bombesin receptor (BnR) family, the lack of known endogenous ligands and high-resolution structure has hindered the understanding of BRS3 signaling and function. We present two cryogenic electron microscopy (cryo-EM) structures of BRS3 in complex with the heterotrimeric Gqprotein in its active states: one bound to the pan-BnR agonist BA1 and the other bound to the synthetic BRS3-specific agonist MK-5046. These structures reveal the architecture of the orthosteric ligand pocket underpinning molecular recognition and provide insights into the structural basis for BRS3’s selectivity and low affinity for bombesin peptides. Examination of conserved micro-switches suggests a shared activation mechanism among BnRs. Our findings shed light on BRS3’s ligand selectivity and signaling mechanisms, paving the way for exploring its therapeutic potential for diabetes, obesity, and related metabolic disorders.

Platelet-activating factor (PAF) is a potent phospholipid mediator crucial in multiple inflammatory and immune responses through binding and activating the PAF receptor (PAFR). However, drug development targeting the PAFR has been limited, partly due to an incomplete understanding of its activation mechanism. Here, we present a 2.9-Å structure of the PAF-bound PAFR-Gicomplex. Structural and mutagenesis analyses unveil a specific binding mode of PAF, with the choline head forming cation-π interactions within PAFR hydrophobic pocket, while the alkyl tail penetrates deeply into an aromatic cleft between TM4 and TM5. Binding of PAF modulates conformational changes in key motifs of PAFR, triggering the outward movement of TM6, TM7, and helix 8 for G protein coupling. Molecular dynamics simulation suggests a membrane-side pathway for PAF entry into PAFR via the TM4-TM5 cavity. By providing molecular insights into PAFR signaling, this work contributes a foundation for developing therapeutic interventions targeting PAF signal axis.

Kisspeptin signaling through its G protein-coupled receptor, KISS1R, plays an indispensable role in regulating reproduction via the hypothalamic-pituitary-gonadal axis. Dysregulation of this pathway underlies severe disorders like infertility and precocious puberty. Here, we present cryo-EM structures of KISS1R bound to the endogenous agonist kisspeptin-10 and a synthetic analog TAK-448. These structures reveal pivotal interactions between peptide ligands and KISS1R extracellular loops for receptor activation. Both peptides exhibit a conserved binding mode, unveiling their common activation mechanism. Intriguingly, KISS1R displays a distinct 40° angular deviation in its intracellular TM6 region compared to other Gq-coupled receptors, enabling distinct interactions with Gq. This study reveals the molecular intricacies governing ligand binding and activation of KISS1R, while highlighting its exceptional ability to couple with Gq. Our findings pave the way for structure-guided design of therapeutics targeting this physiologically indispensable receptor.

Adhesion G-protein-coupled receptors (aGPCRs) are important for organogenesis, neurodevelopment, reproduction and other processes1–6. Many aGPCRs are activated by a conserved internal (tethered) agonist sequence known as the Stachel sequence7–12. Here, we report the cryogenic electron microscopy (cryo-EM) structures of two aGPCRs in complex with Gs: GPR133 and GPR114. The structures indicate that the Stachel sequences of both receptors assume an α-helical–bulge–β-sheet structure and insert into a binding site formed by the transmembrane domain (TMD). A hydrophobic interaction motif (HIM) within the Stachel sequence mediates most of the intramolecular interactions with the TMD. Combined with the cryo-EM structures, biochemical characterization of the HIM motif provides insight into the cross-reactivity and selectivity of the Stachel sequences. Two interconnected mechanisms, the sensing of Stachel sequences by the conserved ‘toggle switch’ W6.53and the constitution of a hydrogen-bond network formed by Q7.49/Y7.49and the P6.47/V6.47φφG6.50motif (φ indicates a hydrophobic residue), are important in Stachel sequence-mediated receptor activation and Gscoupling. Notably, this network stabilizes kink formation in TM helices 6 and 7 (TM6 and TM7, respectively). A common Gs-binding interface is observed between the two aGPCRs, and GPR114 has an extended TM7 that forms unique interactions with Gs. Our structures reveal the detailed mechanisms of aGPCR activation by Stachel sequences and their Gscoupling.

Lysophosphatidylcholine (LPC) is an essential mediator in human lipid metabolism and is associated with a variety of diseases, but the exact identity of LPC receptors remains controversial. Through extensive biochemical and structural analyses, we have identified the orphan receptor GPR119 as the receptor for LPC. The structure of the GPR119–G-protein complex without any added ligands reveals a density map that fits well with LPC, which is further confirmed by mass spectrometry and functional studies. As LPCs are abundant on the cell membrane, their preoccupancy in the receptor may lead to ‘constitutive activity’ of GPR119. The structure of GPR119 bound to APD668, a clinical drug candidate for type 2 diabetes, reveals an exceedingly similar binding mode to LPC. Together, these data highlight structural evidence for LPC function in regulating glucose-dependent insulin secretion through direct binding and activation of GPR119, and provide structural templates for drug design targeting GPR119.

As important drug targets, G protein-coupled receptors (GPCRs) play pivotal roles in a wide range of physiological processes. Extensive efforts of structural biology have been made on the study of GPCRs. However, a large portion of GPCR structures remain unsolved due to structural instability. Recently, AlphaFold2 has been developed to predict structure models of many functionally important proteins including all members of the GPCR family. Herein we evaluated the accuracy of GPCR structure models predicted by AlphaFold2. We revealed that AlphaFold2 could capture the overall backbone features of the receptors. However, the predicted models and experimental structures were different in many aspects including the assembly of the extracellular and transmembrane domains, the shape of the ligand-binding pockets, and the conformation of the transducer-binding interfaces. These differences impeded the use of predicted structure models in the functional study and structure-based drug design of GPCRs, which required reliable high-resolution structural information.

Cholecystokinin receptors, CCKAR and CCKBR, are important neurointestinal peptide hormone receptors and play a vital role in food intake and appetite regulation. Here, we report three crystal structures of the human CCKAR in complex with different ligands, including one peptide agonist and two small-molecule antagonists, as well as two cryo-electron microscopy structures of CCKBR–gastrin in complex with Gi2and Gq, respectively. These structures reveal the recognition pattern of different ligand types and the molecular basis of peptide selectivity in the cholecystokinin receptor family. By comparing receptor structures in different conformational states, a stepwise activation process of cholecystokinin receptors is proposed. Combined with pharmacological data, our results provide atomic details for differential ligand recognition and receptor activation mechanisms. These insights will facilitate the discovery of potential therapeutics targeting cholecystokinin receptors.

Cholecystokinin A receptor (CCKAR) belongs to family A G-protein-coupled receptors and regulates nutrient homeostasis upon stimulation by cholecystokinin (CCK). It is an attractive drug target for gastrointestinal and metabolic diseases. One distinguishing feature of CCKAR is its ability to interact with a sulfated ligand and to couple with divergent G-protein subtypes, including Gs, Giand Gq. However, the basis for G-protein coupling promiscuity and ligand recognition by CCKAR remains unknown. Here, we present three cryo-electron microscopy structures of sulfated CCK-8-activated CCKAR in complex with Gs, Giand Gqheterotrimers, respectively. CCKAR presents a similar conformation in the three structures, whereas conformational differences in the ‘wavy hook’ of the Gα subunits and ICL3 of the receptor serve as determinants in G-protein coupling selectivity. Our findings provide a framework for understanding G-protein coupling promiscuity by CCKAR and uncover the mechanism of receptor recognition by sulfated CCK-8.

Luteinizing hormone and chorionic gonadotropin are glycoprotein hormones that are related to follicle-stimulating hormone and thyroid-stimulating hormone1,2. Luteinizing hormone and chorionic gonadotropin are essential to human reproduction and are important therapeutic drugs3–6. They activate the same G-protein-coupled receptor, luteinizing hormone–choriogonadotropin receptor (LHCGR), by binding to the large extracellular domain3. Here we report four cryo-electron microscopy structures of LHCGR: two structures of the wild-type receptor in the inactive and active states; and two structures of the constitutively active mutated receptor. The active structures are bound to chorionic gonadotropin and the stimulatory G protein (Gs), and one of the structures also contains Org43553, an allosteric agonist7. The structures reveal a distinct ‘push-and-pull’ mechanism of receptor activation, in which the extracellular domain is pushed by the bound hormone and pulled by the extended hinge loop next to the transmembrane domain. A highly conserved 10-residue fragment (P10) from the hinge C-terminal loop at the interface between the extracellular domain and the transmembrane domain functions as a tethered agonist to induce conformational changes in the transmembrane domain and G-protein coupling. Org43553 binds to a pocket of the transmembrane domain and interacts directly with P10, which further stabilizes the active conformation. Together, these structures provide a common model for understanding the signalling of glycoprotein hormone receptors and a basis for drug discovery for endocrine diseases.

Melanocortins are peptide hormones critical for the regulation of stress response, energy homeostasis, inflammation, and skin pigmentation. Their functions are mediated by five G protein-coupled receptors (MC1R–MC5R), predominately through the stimulatory G protein (Gs). MC1R, the founding member of melanocortin receptors, is mainly expressed in melanocytes and is involved in melanogenesis. Dysfunction of MC1R is associated with the development of melanoma and skin cancer. Here we present three cryo-electron microscopy structures of the MC1R–Gs complexes bound to endogenous hormone α-MSH, a marketed drug afamelanotide, and a synthetic agonist SHU9119. These structures reveal the orthosteric binding pocket for the conserved HFRW motif among melanocortins and the crucial role of calcium ion in ligand binding. They also demonstrate the basis of differential activities among different ligands. In addition, unexpected interactions between MC1R and the Gβ subunit were discovered from these structures. Together, our results elucidate a conserved mechanism of calcium-mediated ligand recognition, a specific mode of G protein coupling, and a universal activation pathway of melanocortin receptors.

Bradykinin and kallidin are endogenous kinin peptide hormones that belong to the kallikrein–kinin system and are essential to the regulation of blood pressure, inflammation, coagulation and pain control. Des-Arg10-kallidin, the carboxy-terminal des-Arg metabolite of kallidin, and bradykinin selectively activate two G protein-coupled receptors, type 1 and type 2 bradykinin receptors (B1R and B2R), respectively. The hyperactivation of bradykinin receptors, termed ‘bradykinin storm’, is associated with pulmonary edema in COVID-19 patients, suggesting that bradykinin receptors are important targets for COVID-19 intervention. Here we report two G protein-coupled complex structures of human B1R and B2R bound to des-Arg10-kallidin and bradykinin, respectively. Combined with functional analysis, our structures reveal the mechanism of ligand selectivity and specific activation of the bradykinin receptor. These findings also provide a framework for guiding drug design targeting bradykinin receptors for the treatment of inflammation, cardiovascular disorders and COVID-19.

The acetylcholinesterase (AChE) inhibitors remain key therapeutic drugs for the treatment of Alzheimer’s disease (AD). However, the low-safety window limits their maximum therapeutic benefits. Here, a novel kinetics-driven drug design strategy was employed to discover new-generation AChE inhibitors that possess a longer drug-target residence time and exhibit a larger safety window. After detailed investigations, compound 12was identified as a highly potent, highly selective, orally bioavailable, and brain preferentially distributed AChE inhibitor. Moreover, it significantly ameliorated cognitive impairments in different mouse models with a lower effective dose than donepezil. The X-ray structure of the cocrystal complex provided a precise binding mode between 12and AChE. Besides, the data from the phase I trials demonstrated that 12had good safety, tolerance, and pharmacokinetic profiles at all preset doses in healthy volunteers, providing a solid basis for its further investigation in phase II trials for the treatment of AD.

Adhesion G-protein-coupled receptors (GPCRs) are a major family of GPCRs, but limited knowledge of their ligand regulation or structure is available1–3. Here we report that glucocorticoid stress hormones activate adhesion G-protein-coupled receptor G3 (ADGRG3; also known as GPR97)4–6, a prototypical adhesion GPCR. The cryo-electron microscopy structures of GPR97–Gocomplexes bound to the anti-inflammatory drug beclomethasone or the steroid hormone cortisol revealed that glucocorticoids bind to a pocket within the transmembrane domain. The steroidal core of glucocorticoids is packed against the ‘toggle switch’ residue W6.53, which senses the binding of a ligand and induces activation of the receptor. Active GPR97 uses a quaternary core and HLY motif to fasten the seven-transmembrane bundle and to mediate G protein coupling. The cytoplasmic side of GPR97 has an open cavity, where all three intracellular loops interact with the Goprotein, contributing to the high basal activity of GRP97. Palmitoylation at the cytosolic tail of the Goprotein was found to be essential for efficient engagement with GPR97 but is not observed in other solved GPCR complex structures. Our work provides a structural basis for ligand binding to the seven-transmembrane domain of an adhesion GPCR and subsequent G protein coupling.

Serotonin, or 5-hydroxytryptamine (5-HT), is an important neurotransmitter1,2that activates the largest subtype family of G-protein-coupled receptors3. Drugs that target 5-HT1A, 5-HT1D, 5-HT1Eand other 5-HT receptors are used to treat numerous disorders4. 5-HT receptors have high levels of basal activity and are subject to regulation by lipids, but the structural basis for the lipid regulation and basal activation of these receptors and the pan-agonism of 5-HT remains unclear. Here we report five structures of 5-HT receptor–G-protein complexes: 5-HT1Ain the apo state, bound to 5-HT or bound to the antipsychotic drug aripiprazole; 5-HT1Dbound to 5-HT; and 5-HT1Ein complex with a 5-HT1E- and 5-HT1F-selective agonist, BRL-54443. Notably, the phospholipid phosphatidylinositol 4-phosphate is present at the G-protein–5-HT1Ainterface, and is able to increase 5-HT1A-mediated G-protein activity. The receptor transmembrane domain is surrounded by cholesterol molecules—particularly in the case of 5-HT1A, in which cholesterol molecules are directly involved in shaping the ligand-binding pocket that determines the specificity for aripiprazol. Within the ligand-binding pocket of apo-5-HT1Aare structured water molecules that mimic 5-HT to activate the receptor. Together, our results address a long-standing question of how lipids and water molecules regulate G-protein-coupled receptors, reveal how 5-HT acts as a pan-agonist, and identify the determinants of drug recognition in 5-HT receptors.

Biased signaling of G protein-coupled receptors describes an ability of different ligands that preferentially activate an alternative downstream signaling pathway. In this work, we identified and characterized different N-terminal truncations of endogenous chemokine CCL15 as balanced or biased agonists targeting CCR1, and presented three cryogenic-electron microscopy structures of the CCR1–Gicomplex in the ligand-free form or bound to different CCL15 truncations with a resolution of 2.6–2.9 Å, illustrating the structural basis of natural biased signaling that initiates an inflammation response. Complemented with pharmacological and computational studies, these structures revealed it was the conformational change of Tyr291 (Y2917.43) in CCR1 that triggered its polar network rearrangement in the orthosteric binding pocket and allosterically regulated the activation of β-arrestin signaling. Our structure of CCL15-bound CCR1 also exhibited a critical site for ligand binding distinct from many other chemokine–receptor complexes, providing new insights into the mode of chemokine recognition.

Glucagon-like peptides (GLP-1 and GLP-2) are two proglucagon-derived intestinal hormones that mediate distinct physiological functions through two related receptors (GLP-1R and GLP-2R) which are important drug targets for metabolic disorders and Crohn’s disease, respectively. Despite great progress in GLP-1R structure determination, our understanding on the differences of peptide binding and signal transduction between these two receptors remains elusive. Here we report the electron microscopy structure of the human GLP-2R in complex with GLP-2 and a Gsheterotrimer. To accommodate GLP-2 rather than GLP-1, GLP-2R fine-tunes the conformations of the extracellular parts of transmembrane helices (TMs) 1, 5, 7 and extracellular loop 1 (ECL1). In contrast to GLP-1, the N-terminal histidine of GLP-2 penetrates into the receptor core with a unique orientation. The middle region of GLP-2 engages with TM1 and TM7 more extensively than with ECL2, and the GLP-2 C-terminus closely attaches to ECL1, which is the most protruded among 9 class B G protein-coupled receptors (GPCRs). Functional studies revealed that the above three segments of GLP-2 are essential for GLP-2 recognition and receptor activation, especially the middle region. These results provide new insights into the molecular basis of ligand specificity in class B GPCRs and may facilitate the development of more specific therapeutics.

CpG methylation by de novo DNA methyltransferases (DNMTs) 3A and 3B is essential for mammalian development and differentiation and is frequently dysregulated in cancer1. These two DNMTs preferentially bind to nucleosomes, yet cannot methylate the DNA wrapped around the nucleosome core2, and they favour the methylation of linker DNA at positioned nucleosomes3,4. Here we present the cryo-electron microscopy structure of a ternary complex of catalytically competent DNMT3A2, the catalytically inactive accessory subunit DNMT3B3 and a nucleosome core particle flanked by linker DNA. The catalytic-like domain of the accessory DNMT3B3 binds to the acidic patch of the nucleosome core, which orients the binding of DNMT3A2 to the linker DNA. The steric constraints of this arrangement suggest that nucleosomal DNA must be moved relative to the nucleosome core for de novo methylation to occur.

The polycomb repressive complex 2 (PRC2) catalyzes the methylation of histone H3 on lysine 27 (H3K27) to generate trimethyl-H3K27 (H3K27me3) marks, thereby leading to a repressive chromatin state that inhibits gene expression. C10ORF12 was recently identified as a novel PRC2 interactor. Here, we show that C10ORF12 specifically interacts with PRC2 through its middle region (positions 619–718). C10ORF12 significantly enhances the histone methyltransferase activity of PRC2 in vitro and dramatically increases the total H3K27me3 levels in HeLa cells. C10ORF12 also antagonizes Jarid2, which is an auxiliary factor of the PRC2.2 subcomplex, to promote increased H3K27me3 levels in HeLa cells. Moreover, C10ORF12 alters the substrate preference of PRC2. These results indicate that C10ORF12 functions as a positive regulator of PRC2 and facilitates PRC2-mediated H3K27me3 modification of chromatin. These findings provide new insight into the roles of C10ORF12 in regulating the activity of the PRC2 complex.

Arthrobacter globiformisUricase (AgUricase) is a homotetrameric uricase with the potential for therapeutic use in treating hyperuricemia-related diseases. To achieve sufficient therapeutic effects, it is essential for this enzyme to have high thermostability and long half-life in physiological condition. To improve the thermostability of this enzyme, we introduced a series of cysteine pair mutations into the AgUricase subunits based on its structural model and studied the thermostability of the mutant enzymes with introduced disulfide bridges. Two intersubunit cysteine pair mutations, K12C–E286C and S296C–S296C, were found to markedly increase the melting temperatures of the corresponding mutant enzymes compared with WT AgUricase. The crystal structure of the K12C–E286C mutant at 1.99 Å resolution confirmed the formation of a distinct disulfide bond between the two subunits in the dimer. Structural analysis and biochemical data revealed that the C-terminal loop of AgUricase was flexible, and its interaction with neighboring subunits was required for the stability of the enzyme. We introduced an additional intersubunit K244C–C302 disulfide bond based on the crystal structure of the K12C–E286C mutant and confirmed that this additional disulfide bond further stabilized the flexible C-terminal loop and improved the thermostability of the enzyme. Disulfide cross-linking also protected AgUricase from protease digestion. Our studies suggest that the introduction of disulfide bonds into proteins is a potential strategy for enhancing the thermostability of multimeric proteins for medical applications.

5-HT4R, 5-HT6R, and 5-HT7AR are three constitutively active Gs-coupled 5-HT receptors that have key roles in brain development, learning, memory, cognition, and other physiological processes in the central nervous system. In addition to Gssignaling cascade mediated by these three 5-HT receptors, the ERK1/2 signaling which is dependent on cyclic adenosine monophosphate (cAMP) production and protein kinase A (PKA) activation downstream of Gssignaling has also been widely studied. In this study, we investigated these two signaling pathways originating from the three Gs-coupled 5-HT receptors in AD293 cells. We found that the phosphorylation and activation of ERK1/2 are ligand-induced, in contrast to the constitutively active Gssignaling. This indicates that Gssignaling alone is not sufficient for ERK1/2 activation in these three 5-HT receptors. In addition to Gs, we found that β-arrestin and Fyn are essential for the activation of ERK1/2. Together, these results put forth a novel mechanism for ERK1/2 activation involving the cooperative action of Gs, β-arrestin, and Fyn.