Molecular recognition of morphine and fentanyl by the human µ-opioid receptor.

Morphine and fentanyl are among the most used opioid drugs that confer analgesia and unwanted side effects through both G protein and arrestin signaling pathways of µ-opioid receptor (µOR). Here, we report structures of the human µOR-G protein complexes bound to morphine and fentanyl, which uncover key differences in how they bind the receptor. We also report structures of µOR bound to TRV130, PZM21, and SR17018, which reveal preferential interactions of these agonists with TM3 side of the ligand-binding pocket rather than TM6/7 side. In contrast, morphine and fentanyl form dual interactions with both TM3 and TM6/7 regions. Mutations at the TM6/7 interface abolish arrestin recruitment of µOR promoted by morphine and fentanyl. Ligands designed to reduce TM6/7 interactions display preferential G protein signaling. Our results provide crucial insights into fentanyl recognition and signaling of µOR, which may facilitate rational design of next-generation analgesics.

Dimerization and antidepressant recognition at noradrenaline transporter.

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.

Conserved class B GPCR activation by a biased intracellular agonist.

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.

GPCR activation and GRK2 assembly by a biased intracellular agonist.

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, Gαq and 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 Gαq protein, 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.

Recognition of methamphetamine and other amines by trace amine receptor TAAR1.

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 ß-phenylethylamine (ß-PEA)1 as 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 ß-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.

Hormone- and antibody-mediated activation of the thyrotropin receptor.

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.

Structures of full-length glycoprotein hormone receptor signalling complexes.

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.

ASD2023: towards the integrating landscapes of allosteric knowledgebase.

Allosteric regulation, induced by perturbations at an allosteric site topographically distinct from the orthosteric site, is one of the most direct and efficient ways to fine-tune macromolecular function. The Allosteric Database (ASD; accessible online at http://mdl.shsmu.edu.cn/ASD) has been systematically developed since 2009 to provide comprehensive information on allosteric regulation. In recent years, allostery has seen sustained growth and wide-ranging applications in life sciences, from basic research to new therapeutics development, while also elucidating emerging obstacles across allosteric research stages. To overcome these challenges and maintain high-quality data center services, novel features were curated in the ASD2023 update: (i) 66 589 potential allosteric sites, covering > 80% of the human proteome and constituting the human allosteric pocketome; (ii) 748 allosteric protein-protein interaction (PPI) modulators with clear mechanisms, aiding protein machine studies and PPI-targeted drug discovery; (iii) 'Allosteric Hit-to-Lead,' a pioneering dataset providing panoramic views from 87 well-defined allosteric hits to 6565 leads and (iv) 456 dualsteric modulators for exploring the simultaneous regulation of allosteric and orthosteric sites. Meanwhile, ASD2023 maintains a significant growth of foundational allosteric data. Based on these efforts, the allosteric knowledgebase is progressively evolving towards an integrated landscape, facilitating advancements in allosteric target identification, mechanistic exploration and drug discovery.

Revealing the signaling of complement receptors C3aR and C5aR1 by anaphylatoxins.

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.

Ligand recognition and G-protein coupling selectivity of cholecystokinin A receptor.

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, Gi and 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, Gi and Gq heterotrimers, 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.

Loops Mediate Agonist-Induced Activation of the Stimulator of Interferon Genes Protein.

The stimulator of interferon genes (STING) is an important therapeutic target for cancer diseases. The activated STING recruits downstream tank-binding kinase 1 (TBK1) to trigger several important immune responses. However, the molecular mechanism of how agonist molecules mediate the STING-TBK1 interactions remains elusive. Here, we performed molecular dynamics simulations to capture the conformational changes of STING and TBK1 upon agonist binding. Our simulations revealed that multiple helices (α5-α7) and especially three loops (loop 6, loop 8, and C-terminal tail) of STING participated in the allosteric mediation of the STING-TBK1 interactions. Consistent results were also observed in the simulations of the constitutive activating mutant of STING (R284S). We further identified α5 as a key region in this agonist-induced activation mechanism of STING. Free-energy perturbation calculations of multiple STING agonists demonstrated that an alkynyl group targeting α5 is a determinant for agonist activities. These results not only offer deeper insights into the agonist-induced allosteric mediation of STING-TKB1 interactions but also provide a guidance for future drug development of this important therapeutic target.

Discovery of a novel GPR35 agonist with high and equipotent species potency for oral treatment of IBD.

The G protein-coupled receptor 35 (GPR35) has been identified as a potential target in the treatment of inflammatory bowel disease (IBD). However, the lack of high and equipotent agonists on both human and mouse GPR35 has limited the in vivo study of GPR35 agonists in mouse models of IBD. In this study, structural modifications to lodoxamide provides a series of high and equivalent agonists on human, mouse, and rat GPR35. These molecules eliminate the species selectivity of human to mouse and rat orthologs that have been prevalent with GPR35 agonists including lodoxamide. The cLogP properties are also optimized to make the compounds more obedient to drug-like rules, yielding compound 4b (cLogP = 2.41), which activates human, mouse or rat GPR35 with EC50 values of 76.0, 63.7 and 77.8 nM, respectively. Oral administration of compound 4b at 20 mg/kg alleviates clinical symptoms of DSS-induced IBD in mice, and is slightly more effective than 5-ASA at 200 mg/kg. In summary, it can serve as a new start point for exploiting more potent GPR35 agonists without species differences for the treatment of IBD, and warrants further study.

AlphaFold2 versus experimental structures: evaluation on G protein-coupled receptors.

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.

Molecular recognition of two endogenous hormones by the human parathyroid hormone receptor-1.

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.

Improving machine learning force fields for molecular dynamics simulations with fine-grained force metrics.

Machine learning force fields (MLFFs) have gained popularity in recent years as they provide a cost-effective alternative to ab initio molecular dynamics (MD) simulations. Despite a small error on the test set, MLFFs inherently suffer from generalization and robustness issues during MD simulations. To alleviate these issues, we propose global force metrics and fine-grained metrics from element and conformation aspects to systematically measure MLFFs for every atom and every conformation of molecules. We selected three state-of-the-art MLFFs (ET, NequIP, and ViSNet) and comprehensively evaluated on aspirin, Ac-Ala3-NHMe, and Chignolin MD datasets with the number of atoms ranging from 21 to 166. Driven by the trained MLFFs on these molecules, we performed MD simulations from different initial conformations, analyzed the relationship between the force metrics and the stability of simulation trajectories, and investigated the reason for collapsed simulations. Finally, the performance of MLFFs and the stability of MD simulations can be further improved guided by the proposed force metrics for model training, specifically training MLFF models with these force metrics as loss functions, fine-tuning by reweighting samples in the original dataset, and continued training by recruiting additional unexplored data.

IDDB: a comprehensive resource featuring genes, variants and characteristics associated with infertility.

Infertility is a complex multifactorial disease that affects up to 10% of couples across the world. However, many mechanisms of infertility remain unclear due to the lack of studies based on systematic knowledge, leading to ineffective treatment and/or transmission of genetic defects to offspring. Here, we developed an infertility disease database to provide a comprehensive resource featuring various factors involved in infertility. Features in the current IDDB version were manually curated as follows: (i) a total of 307 infertility-associated genes in human and 1348 genes associated with reproductive disorder in 9 model organisms; (ii) a total of 202 chromosomal abnormalities leading to human infertility, including aneuploidies and structural variants; and (iii) a total of 2078 pathogenic variants from infertility patients' samples across 60 different diseases causing infertility. Additionally, the characteristics of clinically diagnosed infertility patients (i.e. causative variants, laboratory indexes and clinical manifestations) were collected. To the best of our knowledge, the IDDB is the first infertility database serving as a systematic resource for biologists to decipher infertility mechanisms and for clinicians to achieve better diagnosis/treatment of patients from disease phenotype to genetic factors. The IDDB is freely available at http://mdl.shsmu.edu.cn/IDDB/.

Computational characterization of transducer recognition of ß2 adrenergic receptor.

As an important drug target, ß2 adrenergic receptor (B2AR) regulates many physiological processes, including cardiac function, airway tone and metabolic functions. The selective coupling between B2AR and specific transducers is critical for the physiological action of the receptor. However, the molecular mechanism by which B2AR recognizes different transducers remains elusive. Here, molecular dynamics simulations of B2AR binding to three functionally important transducers (Gs, Gi and ß-arrestin 1) unveiled distinct binding modes of the receptor. Involving transmembrane helices TMs 2-7 and intracellular loops ICLs 2-3, different binding interfaces for Gs and ß-arrestin 1 were identified in the simulation models and further validated by various assays. The distinct recognition mode of B2AR for Gi was computationally characterized. Insights into receptor-transducer communication not only enhance our understanding of signaling bias, but also offer hints for rational drug design targeting specific signaling pathways of G-protein coupled receptors (GPCRs).

Unraveling allosteric landscapes of allosterome with ASD.

Allosteric regulation is one of the most direct and efficient ways to fine-tune protein function; it is induced by the binding of a ligand at an allosteric site that is topographically distinct from an orthosteric site. The Allosteric Database (ASD, available online at http://mdl.shsmu.edu.cn/ASD) was developed ten years ago to provide comprehensive information related to allosteric regulation. In recent years, allosteric regulation has received great attention in biological research, bioengineering, and drug discovery, leading to the emergence of entire allosteric landscapes as allosteromes. To facilitate research from the perspective of the allosterome, in ASD 2019, novel features were curated as follows: (i) >10 000 potential allosteric sites of human proteins were deposited for allosteric drug discovery; (ii) 7 human allosterome maps, including protease and ion channel maps, were built to reveal allosteric evolution within families; (iii) 1312 somatic missense mutations at allosteric sites were collected from patient samples from 33 cancer types and (iv) 1493 pharmacophores extracted from allosteric sites were provided for modulator screening. Over the past ten years, the ASD has become a central resource for studying allosteric regulation and will play more important roles in both target identification and allosteric drug discovery in the future.

Conformational Selection Mechanism Provides Structural Insights into the Optimization of APC-Asef Inhibitors.

Metastasis is the major cause of death in colorectal cancer and it has been proven that inhibiting an interaction between adenomatous polyposis coli (APC) and Rho guanine nucleotide exchange factor 4 (Asef) efficaciously restrain metastasis. However, current inhibitors cannot achieve a satisfying effect in vivo and need to be optimized. In the present study, we applied molecular dynamics (MD) simulations and extensive analyses to apo and holo APC systems in order to reveal the inhibitor mechanism in detail and provide insights into optimization. MD simulations suggested that apo APC takes on a broad array of conformations and inhibitors stabilize conformation selectively. Representative structures in trajectories show specific APC-ligand interactions, explaining the different binding process. The stability and dynamic properties of systems elucidate the inherent factors of the conformation selection mechanism. Binding free energy analysis quantitatively confirms key interface residues and guide optimization. This study elucidates the conformation selection mechanism in APC-Asef inhibition and provides insights into peptide-based drug design.

Characteristics of Allosteric Proteins, Sites, and Modulators.

Allostery is considered one of the most direct and efficient ways to regulate biological macromolecule functions. Allostery is increasingly receiving attention in the field of drug discovery because of the unique advantages of allosteric modulators such as high selectivity and low toxicity. Because of technical breakthroughs in the allosteric studies, the understanding of the characteristics of allosteric entities such as allosteric proteins and their allosteric sites and modulators has made great strides. These features play a critical role in both the evolution of the allosteric concept and the prediction of allosteric interactions. In this chapter, we highlight the fundamental characteristics of allosteric proteins, allosteric sites, and allosteric modulators. Importantly, the applications of such principles in real cases are depicted in detail. Collectively, these characteristics are beneficial in aiding allosteric drug design and allosteric mechanism research.