Biology subjects

Xu, H. E.

Publications and source records attributed to Xu, H. E..

At least 19 recordsLinked to original sources

bioRxiv2026

Fine-tuning an intracellular allosteric pocket programs GPCR signaling bias

Biased signaling of G protein-coupled receptors (GPCRs) offers a route for precise therapeutics with fewer adverse effects. Intracellular allosteric ligands can impose such bias, yet how subtle chemical changes within these pockets reshape receptor-transducer coupling and downstream signaling remains unclear. Here, we systematically fine-tuned the intracellular allosteric pocket of neurotensin receptor 1 (NTSR1) through chemical modification of the {beta}-arrestin-biased ligand SBI-553. Minor changes to the ligand scaffold altered transducer-coupling preferences, generating distinct profiles of G-protein and {beta}-arrestin signaling. Cryo-electron microscopy structures of NTSR1-{beta}-arrestin2 complexes bound to designed allosteric probes revealed ligand-dependent remodeling of the intracellular cavity that reshapes the receptor-effector interface. Alanine scanning identified transducer-sensitive residues within and beyond the immediate ligand-contact region. In mice, SBI-553 and N065 produced distinct physiological profiles, with N065 improving survival in a lethal endotoxemia model. Finally, phosphoproteomic profiling revealed partially overlapping but distinct downstream signaling profiles among the three modulators. Together, these findings establish the intracellular allosteric pocket as a tunable determinant of transducer coupling and show that closely related modulators can generate distinct signaling and physiological profiles.

pharmacology and toxicology↗

bioRxiv2026

Cooperative Peripheral CB1 Inhibition by Extracellular Antibody Binding and Orthosteric Antagonism

The cannabinoid receptor CB1 is an important therapeutic target for obesity and metabolic disorders, yet centrally acting CB1 antagonists are limited by neuropsychiatric side effects. Although the anti-CB1 antibody Nimacimab has advanced to Phase 2 clinical trials for metabolic disorders, the molecular mechanisms underlying peripheral CB1 inhibition remain poorly understood. Here, we report cryo-EM structures of human CB1 bound to Nimacimab alone and in complex with the orthosteric antagonists Rimonabant and JD5037. Unlike previously characterized inhibitory GPCR antibodies that block ligand access through steric occlusion or directly engage orthosteric sites, Nimacimab functions through extracellular allosteric inhibition by stabilizing an inactive-like extracellular architecture of CB1. Antibody binding restricts extracellular conformational dynamics and allosterically stabilizes inactive-state signaling microswitches. Importantly, extracellular antibody engagement is structurally compatible with orthosteric antagonism and cooperatively enhances CB1 inhibition. Structural analysis further reveals how JD5037 remodels the extracellular vestibule to achieve peripheral-selective antagonism. Together, these findings establish extracellular allosteric inhibition as a distinct mechanism of GPCR regulation and provide a structural framework for the development of peripherally selective CB1 therapeutics.

pharmacology and toxicology↗

bioRxiv2026

β-arrestin1 directly engages Gαs to sustain endosomal GPCR signaling

{beta}-arrestins ({beta}arrs) are canonical terminators of G protein-coupled receptor (GPCR) signaling, yet internalized receptors paradoxically sustain {beta}arr-dependent Gs/cAMP signaling from endosomes. How a desensitizing scaffold can instead promote G protein activation has remained unresolved, because the receptor-{beta}arr-G protein assemblies described so far position the two transducers on opposite faces of the receptor, with no contact between them. Using the parathyroid hormone type 1 receptor (PTH1R), we show that {beta}arr1 binds Gs directly, engages its nucleotide-free state, and does so far more strongly than it engages G{beta}{gamma}. A cryo-EM structure of the agonist-bound PTH1R-{beta}arr1-Gs complex at ~3.1 &Aring resolution reveals how: {beta}arr1 anchors to the phosphorylated receptor C-tail, leaving the transmembrane core free for Gs, and reorients to build a direct, three-node interface in which its finger, C- and back loops clamp the Switch I, Switch II and 3/{beta}5 regions of Gs. Disrupting either side of this interface abolishes complex assembly and selectively collapses the internalization-dependent phase of cAMP production, and the same interface governs {beta}arr1-Gs coupling at the glucagon-like peptide-1 receptor. {beta}-arrestin1 therefore acts as a positive regulator that holds Gs in a signaling-competent state, providing a unifying structural basis for sustained endosomal GPCR signaling.

biochemistry↗

bioRxiv2026

Conservation of Nuclear Receptor DAF-12 with Evidence for Ligand Diversification in Plant-Parasitic Nematodes

Plant-parasitic nematodes cause extensive crop losses that threaten global food security and create substantial economic hardship, yet strategies for their control remain limited. Although plant-parasitic nematodes have life cycles that mirror those of their free-living and animal-parasitic relatives, comparatively little is known about the signals that drive life-cycle progression. Dafachronic acids (DAs) are steroid hormones that control life cycle by activating DAF-12 nuclear receptors in Caenorhabditis elegans and animal-parasitic nematodes. To define whether an analogous pathway exists in plant-parasitic nematodes, we investigated the responsiveness of DAF-12 orthologs from six plant-parasitic nematode species to {Delta}7-DA, an endogenous DAF-12 ligand in C. elegans and several animal-parasitic nematodes. We find that whereas some plant-parasitic nematode receptors are activated by {Delta}7-DA, others are not. Structural modeling of all six ligand-binding domains, together with docking of {Delta}7-DA into each model, indicates that responsiveness is governed by a combinatorial ligand-recognition network surrounding the ligand carboxylate rather than by any single contact residue. We further demonstrate that egg and second-stage juvenile extracts from the soybean cyst nematode, Heterodera glycines, lack detectable DA but contain distinct lipophilic fractions that activate its DAF-12 ortholog. Together, these findings suggest that many plant-parasitic nematodes utilize endogenous DAF-12 ligands that are structurally distinct from DA. Uncovering modulators of this pathway could lead to new strategies to combat nematode-driven crop loss.

molecular biology↗

bioRxiv2026

Molecular principles for graded activation of dopamine D1 receptor

G protein-coupled receptors (GPCRs) signal across a continuum of activation states, yet how ligands encode distinct signaling efficacies remains poorly understood. Here, we define the molecular mechanism underlying graded activation of the dopamine D1 receptor (D1R), a major therapeutic target for neuropsychiatric disorders. Functional analyses reveal that two widely used pharmacological tools, LE300 and SCH23390, possess intrinsic efficacy as an inverse agonist and a weak partial agonist, respectively, rather than the efficacy-silent neutral antagonists. Structural, molecular dynamics and mutagenesis analyses capture previously unrecognized inactive and intermediate receptor activation states that bridge known active conformations, and reveal that ligand efficacy is encoded through the progressive engagement of a conserved activation pathway centered on the W6.48 toggle switch. Guided by this mechanism, a single chemical modification markedly increases the agonist efficacy of SCH23390. Comparison with dopamine D2 receptor structures further reveals a conserved mechanism of inverse agonism despite distinct subtype-specific recognition. Together, these findings establish a structural framework for graded agonism at D1R and provide general principles for the rational design of efficacy-tuned therapeutics at dopamine receptors and related GPCRs.

biochemistry↗

bioRxiv2026

Accurate de novo design of peptides from programming biophysical landscape

Peptides regulate virtually every cellular process and emerge as transformative modalities in bioengineering and therapeutics. However, the de novo design of functional peptides remains challenging, as peptide-protein interactions are governed by delicate and dynamic biophysics that are difficult to model, and experimental datasets are scarce. Here we present NeoPep, a generative deep-learning framework that encodes biophysical principles to accurately design functional peptides de novo. By integrating over 5 million peptide-protein complexes spanning experimentally determined, sequence mimics, and structure ensembles, NeoPep learns the complex biophysical landscape governing peptide function and enables its programmable control. In prospective application across 10 diverse and challenging targets, NeoPep generated potent peptide binders, agonists, and antagonists with hit rates of 12.5-66.7%, even in the absence of defined binding sites or structure information. Beyond de novo co-design, NeoPep supports standalone structure or sequence redesign, readily discriminating subtle context differences. In the structure redesign mode, it generates highly selective peptides with atomic-level conformational accuracy (C RMSD < 2.0 [A] to our solved cryo-EM structure). This structural precision circumvents the need for experimental structure determination, further accelerating iterative sequence redesign to yield a 43.3-fold improvement in potency. These findings establish a general framework for translating biophysical principles into actionable peptide functions, with broad implications for basic research, bioengineering, and medicine.

molecular biology↗

bioRxiv2025

Multiple Allosteric Sites Allow for Synergistic Enhancement of GPCR Signaling

Allosteric modulation of G protein-coupled receptors (GPCRs) is an emerging therapeutic paradigm that has proven effective, yet the cooperative action of multiple modulators remains unexplored. Here, we reveal how positive allosteric modulators (PAMs) synergistically enable extraordinary signal amplification through the D1 dopamine receptor (D1R). We developed UNC9815 and UNC10062 as enhanced D1R PAMs from the parent compound MLS6585, and then employed cryo-electron microscopy to reveal concurrent occupancy of three distinct allosteric sites by PAMs of different scaffolds, including LY3154207, BMS-A1, and our UNC compounds. Remarkably, we discovered two adjacent allosteric pockets at the transmembrane helix (TM) 1-7 interface: BMS-A1 occupies an intracellular site promoting activation through TM7 conformational shifts, while the UNC compounds complementarily stabilize the extracellular side of the interface. When combined with LY3154207, this cooperative architecture enhances dopamine potency by more than 1,000-fold. These findings elucidate the first structural blueprint for multi-site GPCR cooperativity, unlocking transformative therapeutic strategies inaccessible to orthosteric and single- site allosteric drugs.

molecular biology↗

bioRxiv2025

Structure based discovery of antipsychotic-like TAAR1 agonists

Schizophrenia is a severe mental illness whose current treatments primarily target dopamine and serotonin receptors. These drugs often cause side effects and vary in effectiveness across patients. The trace amine-associated receptor 1 (TAAR1), which modulates monoamine signaling, has emerged as a promising alternative target. To discover new TAAR1 ligands, we computationally docked 65 million molecules against the active state of TAAR1 and experimentally tested 55 of those highly ranked. Fourteen molecules active against TAAR1 with potencies ranging from mid-nanomolar to micromolar, all as agonists. This high functional selectivity may reflect the compact conformation adopted by the activated TAAR1 orthosteric site. While this was favorable for agonist prioritization, simulations suggest that it can be over-optimized for initial hit rates at the expense of subsequent affinity maturation. Here, hit optimization yielded nanomolar agonists whose docking-predicted poses were confirmed by cryo-EM. Three agonists had high brain exposure and potencies apparently better than the investigational drug ulotaront and sufficient for behavioral studies. All three potently normalized amphetamine-induced pre-pulse inhibition in mice, a model for schizophrenia, without catalepsy, a common side effect of traditional antipsychotics.

biochemistry↗

bioRxiv2025

Molecular architecture of OXGR1 reveals evolutionary conserved mechanisms for metabolite surveillance

The ability of cells to sense and respond to metabolic signals is fundamental to life, yet the molecular mechanisms underlying metabolite surveillance remain incompletely understood. Here, we elucidate the structural basis of metabolite recognition by OXGR1, a GPCR that monitors key intermediates in the tricarboxylic acid cycle (TCA). Using cryo-electron microscopy, we determined four cryo-EM structures of OXGR1 bound to -ketoglutarate (AKG), itaconate (ITA), and structural related succinate (SUC) and maleate (MA). These structures reveal a positively charged binding pocket and an extensive hydrogen-bond network critical for OXGR1 recognizing dicarboxylic acids. In addition, we identify a distinct arrangement of hydrophobic residues that modulates ligand potency and selectivity. Mutational analysis and molecular dynamics simulations further demonstrate that non-canonical micro-switch motifs, including FRY and NLxxY, are essential for ligand recognition and receptor activation. Comparative structural and evolutionary analyses indicate that these mechanisms are conserved across species, underscoring the critical role of OXGR1 in maintaining metabolic homeostasis. Together, our findings provide a mechanistic framework for metabolite sensing via OXGR1 and suggest potential strategies for therapeutic modulation of metabolic and inflammatory diseases.

molecular biology↗

bioRxiv2025

Molecular Mechanism of pH Sensing and Activation in GPR4 Reveals Proton-Mediated GPCR Signaling

Maintaining pH homeostasis is critical for cellular function across all living organisms. Proton-sensing G protein-coupled receptors (GPCRs), particularly GPR4, play a pivotal role in cellular responses to pH changes, yet the molecular mechanisms underlying their proton sensing and activation remain incompletely understood. Here we present high-resolution cryo-electron microscopy structures of GPR4 in complex with G proteins under physiological and acidic pH conditions. Our structures expose an intricate proton-sensing mechanism driven by a sophisticated histidine network in the receptors extracellular domain. Upon protonation of key histidines under acidic conditions, a remarkable conformational cascade is initiated, propagating from the extracellular region to the intracellular G protein-coupling interface. This dynamic process involves precise transmembrane helix rearrangements and conformational shifts of conserved motifs, mediated by strategically positioned water molecules. Notably, we discovered a bound bioactive lipid, lysophosphatidylcholine, which has positive allosteric effects on GPR4 activation. These findings provide a comprehensive framework for understanding proton sensing in GPCRs and the interplay between pH sensing and lipid regulation, offering insights into cellular pH homeostasis and potential therapies for pH-related disorders.

molecular biology↗

bioRxiv2024

Structural basis of augment taurine uptake by taurine transporter alleviating cellular senescence

Taurine, a sulfur-containing amino acid, is critical for diverse physiological processes including liver function, cardiovascular health, and neurological development. Its deficiency has been linked to liver damage, cardiomyopathy, retinal degeneration, and accelerated aging. Despite its importance in metabolic regulation and disease prevention, the structural basis for taurines cellular uptake and its potential therapeutic applications have remained elusive. Here, we present high-resolution cryo-electron microscopy structures of the human taurine transporter (TauT) in multiple conformational states, revealing key insights into the taurine transport mechanism. We identify five distinct taurine-binding sites, elucidating a continuous pathway for taurine translocation across the cell membrane. Notably, we discover a novel gating mechanism involving a conserved salt bridge, which when disrupted, significantly enhances taurine uptake. Leveraging these structural insights, we demonstrate that augmented taurine uptake via TauT alleviates stress-induced senescence in biliary epithelial cells, potentially through the upregulation of the stress-responsive gene ATF3. Our findings not only advance the understanding of neurotransmitter transport but also unveil TauT as a promising therapeutic target for age-related liver diseases and other senescence-associated disorders. This work bridges structural biology with physiological function, offering new avenues for developing interventions to combat cellular aging and taurine-deficiency related diseases.

biochemistry↗

bioRxiv2024

Structural Basis of GPCR-G Protein Pre-coupling and Activation: Insights from CCR1-Gi Complex

GPCR-mediated G protein activation cycle contains obligated step of the receptor-G protein assembly before G protein activation (pre-coupling state). However, transient nature of this pre-coupling state has prevented structural and mechanistic understanding of this essential step of G protein activation cycle. In this study, we discover that CCR1 has high level of pre-coupling state, which allows its rapid response to chemokines. Taking advantage of this observation, we uncover molecular mechanism of the pre-coupling state by solving the cryo-electron microscopy structure of the chemokine receptor CCR1 in complex with its cognate G protein (Gi) in a pre-coupled, inactivated state. This structure reveals that CCR1 adopts a conformation distinct from both its fully active and inactive states, with the G proteins 5 helix partially inserted into the receptors intracellular cavity. Notably, the C-terminal four residues of the G subunit are disordered in this pre-coupled state, contrasting with their well-defined -helical structure in the fully active complex. Functional assays demonstrate that while deletion of these four C-terminal G residues does not affect pre-coupling, it abolishes G protein activation upon agonist binding. This finding highlights the critical role of these residues in GPCR-mediated G protein activation, but not in initial recruitment. Furthermore, our studies indicate that the ability to form pre-coupled complexes is conserved among chemokine receptors, suggesting a common mechanism for rapid signal transduction in this GPCR subfamily. These results provide the first structural evidence for GPCR-G protein pre-coupling and offer molecular insights into the transition from inactive to active states. Our findings fill the long-missing gap in understanding GPCR-mediated G protein activated cycle.

biophysics↗

bioRxiv2024

Structural Insights into Selective and Dual Antagonism of EP2 and EP4 Prostaglandin Receptors

Prostaglandin E2 (PGE2) signaling through EP2 and EP4 receptors plays a crucial role in inflammation, pain, and cancer progression. Selective and dual antagonists of these receptors have shown promising therapeutic potential. However, the structural basis for their binding modes and selectivity remained unclear. Here, we present cryo-EM structures of human EP2 and EP4 receptors in complex with selective antagonists PF-04418948 and grapiprant, respectively, as well as the dual antagonist TG6-129. These structures reveal distinct binding pockets and interaction networks that govern antagonist selectivity and efficacy. Combining the structural data and functional studies, we uncover the key residues and structural features that differentiate EP2 and EP4 binding sites, including a unique {pi}-{pi} stacking interaction in EP2 and variations in pocket shape and charge distribution. The dual antagonist TG6-129 exhibited a novel binding mode, interacting deeply with EP2 but only shallowly with EP4 in a two-warhead manner. These findings provide a structural framework for understanding prostanoid receptor pharmacology and offer valuable insights for the rational design of improved selective and dual antagonists targeting EP2 and EP4 receptors.

biochemistry↗

bioRxiv2024

Molecular mechanisms of uric acid transport by the native human URAT1 and its inhibition by anti-gout drugs

Gout, a common and painful disease, stems from hyperuricemia, where elevated blood uric acid levels lead to urate crystal formation in joints and kidneys. The human urate transporter 1 (hURAT1) plays a critical role in urate homeostasis by facilitating urate reabsorption in the renal proximal tubule, making it a key target for gout therapy. Pharmacological inhibition of hURAT1 with drugs such as dotinurad, benzbromarone, lesinurad, and verinurad promotes uric acid excretion and alleviates gout symptoms. Here we present cryo-electron microscopy structures of native hURAT1 bound with these anti-gout drugs in the inward-open state, and with uric acid in inward-open, outward-open, and occluded states. Complemented by mutagenesis and cell-based assays, these structures reveal the mechanisms of uric acid reabsorption and hURAT1 inhibition. Our findings elucidate the molecular basis of uric acid transport and anti-gout medication action, and provide a structural framework for the rational design of next-generation therapies for hyperuricemia and gout.

biochemistry↗

bioRxiv2023

Structure basis for recognition of 26RFa by the pyroglutamylated RFamide peptide receptor

The neuropeptide 26RFa, a member of the RF-amide peptide family, activates the pyroglutamylated RF-amide peptide receptor (QRFPR), a class A GPCR. The 26RFa/QRFPR system plays critical roles in energy homeostasis, making QRFPR an attractive drug target for treating obesity, diabetes, and eating disorders. However, the lack of structural information has hindered our understanding of the peptide recognition and regulatory mechanism of QRFPR, impeding drug design efforts. In this study, we determined the cryo-EM structure of the Gq-coupled QRFPR bound to 26RFa. The structure reveals a unique assembly mode of the receptor extracellular regions and the peptide N-terminus and elucidates the recognition mechanism of the C-terminal heptapeptide of 26RFa within the transmembrane binding pocket of QRFPR. The study also clarifies the similarities and distinctions in the binding pattern of the RF-amide moiety in five RF-amide peptides and the RY-amide segment in neuropeptide Y. These findings deepen our understanding of the RF-amide peptides recognition, aiding in the rational design of drugs targeting QRFPR and other RF-amide receptors.

biophysics↗

bioRxiv2023

Mechanisms of ligand recognition and activation of melanin-concentrating hormone receptors

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide that regulates food intake, energy balance, and other physiological functions by stimulating MCHR1 and MCHR2 receptors, both of which are class A G protein-coupled receptors. MCHR1 can couple with multiple G-proteins, including Gi/o, Gq/11, and Gs, while MCHR2 only couple to Gq/11. Here we present cryo-electron microscopy structures of MCH-activated MCHR1 with Gi and MCH-activated MCHR2 with Gq complexes, at the global resolutions of 3.01 [A] and 2.40 [A], respectively. These structures reveal that MCH adopts a consistent cysteine-mediated hairpin loop configuration in both receptors. A central arginine from the LGRVY core motif between the two cysteines of MCH penetrates deeply into the transmembrane pocket, triggering receptor activation. Integrated with mutational and functional insights, our findings elucidate the molecular underpinnings of ligand recognition and MCH receptor activation, offering a structured foundation for targeted drug design.

molecular biology↗

bioRxiv2023

A conserved and druggable pocket in class B G protein coupled receptors for orally active small molecule agonists

Class B G protein-coupled receptors (GPCRs), including glucagon-like receptor 1 (GLP-1R) and parathyroid hormone receptor 1 (PTH1R), are peptide hormone receptors and important drug targets. Injectable peptide drugs targeting class B GPCRs have been developed for the treatment of many diseases, including type 2 diabetes, obesity, and osteoporosis, but orally available small molecule drugs are hotly pursued in the field, especially small molecule agonists of GLP-1R and PTH1R. Here we report the first high-resolution structure of the 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 interface of PTH1R and Gs. The binding site of PCO371 is totally different from all binding sites previously reported for small molecules or peptide ligands in GPCRs. Residues that make up the PCO371 binding pocket are mostly conserved in class B GPCRs and a single mutation in PTH type 2 receptor (PTH2R) and two residue mutations in GLP-1R convert these receptors to respond to PCO371 activation. Functional assays reveal that PCO371 is a G-protein biased agonist that is defective in promoting PTH1R-mediated arrestin signaling. Together, these results uncover a distinct binding site for designing small molecule agonists for PTH1R and possible other members of class B GPCRs and define a receptor conformation that is only specific for G protein activation but not arrestin signaling. These insights should facilitate the design of distinct types of class B GPCR small molecule agonists for various therapeutic indications.

biochemistry↗

bioRxiv2023

Structural basis of peptide recognition and activation of endothelin receptors

Endothelin system comprises three endogenous 21-amino-acid peptide ligands endothelin-1, -2, and -3 (ET-1/2/3), and two G protein-coupled receptor (GPCR) subtypes--endothelin receptor A (ETAR) and B (ETBR). Since ET-1, the first endothelin, was identified in 1988 as one of the most potent endothelial cell-derived vasoconstrictor peptides with long-lasting actions, the endothelin system has attracted extensive attention due to its critical role in vasoregulation and close relevance in cardiovascular-related diseases. Here we present three cryo-electron microscopy structures of ETAR and ETBR bound to ET-1 and ETBR bound to the selective peptide IRL1620. These structures reveal a highly conserved recognition mode of ET-1 and characterize the ligand selectivity by ETRs. They also present several conformation features of the active ETRs, thus revealing a specific activation mechanism. Together, these findings deepen our understanding of endothelin system regulation and offer an opportunity to design selective drugs targeting specific ETR subtypes.

molecular biology↗