Structure-based design of non-hypertrophic apelin receptor modulator.

Apelin is a key hormone in cardiovascular homeostasis that activates the apelin receptor (APLNR), which is regarded as a promising therapeutic target for cardiovascular disease. However, adverse effects through the ß-arrestin pathway limit its pharmacological use. Here, we report cryoelectron microscopy (cryo-EM) structures of APLNR-Gi1 complexes bound to three agonists with divergent signaling profiles. Combined with functional assays, we have identified "twin hotspots" in APLNR as key determinants for signaling bias, guiding the rational design of two exclusive G-protein-biased agonists WN353 and WN561. Cryo-EM structures of WN353- and WN561-stimulated APLNR-G protein complexes further confirm that the designed ligands adopt the desired poses. Pathophysiological experiments have provided evidence that WN561 demonstrates superior therapeutic effects against cardiac hypertrophy and reduced adverse effects compared with the established APLNR agonists. In summary, our designed APLNR modulator may facilitate the development of next-generation cardiovascular medications.

Conformational transitions and activation of the adhesion receptor CD97.

Adhesion G protein-coupled receptors (aGPCRs) are evolutionarily ancient receptors involved in a variety of physiological and pathophysiological processes. Modulators of aGPCR, particularly antagonists, hold therapeutic promise for diseases like cancer and immune and neurological disorders. Hindered by the inactive state structural information, our understanding of antagonist development and aGPCR activation faces challenges. Here, we report the cryo-electron microscopy structures of human CD97, a prototypical aGPCR that plays crucial roles in immune system, in its inactive apo and G13-bound fully active states. Compared with other family GPCRs, CD97 adopts a compact inactive conformation with a constrained ligand pocket. Activation induces significant conformational changes for both extracellular and intracellular sides, creating larger cavities for Stachel sequence binding and G13 engagement. Integrated with functional and metadynamics analyses, our study provides significant mechanistic insights into the activation and signaling of aGPCRs, paving the way for future drug discovery efforts.

Structural basis of peptidomimetic agonism revealed by small- molecule GLP-1R agonists Boc5 and WB4-24.

Glucagon-like peptide-1 receptor (GLP-1R) agonists are effective in treating type 2 diabetes and obesity with proven cardiovascular benefits. However, most of these agonists are peptides and require subcutaneous injection except for orally available semaglutide. Boc5 was identified as the first orthosteric nonpeptidic agonist of GLP-1R that mimics a broad spectrum of bioactivities of GLP-1 in vitro and in vivo. Here, we report the cryoelectron microscopy structures of Boc5 and its analog WB4-24 in complex with the human GLP-1R and Gs protein. Bound to the extracellular domain, extracellular loop 2, and transmembrane (TM) helices 1, 2, 3, and 7, one arm of both compounds was inserted deeply into the bottom of the orthosteric binding pocket that is usually accessible by peptidic agonists, thereby partially overlapping with the residues A8 to D15 in GLP-1. The other three arms, meanwhile, extended to the TM1-TM7, TM1-TM2, and TM2-TM3 clefts, showing an interaction feature substantially similar to the previously known small-molecule agonist LY3502970. Such a unique binding mode creates a distinct conformation that confers both peptidomimetic agonism and biased signaling induced by nonpeptidic modulators at GLP-1R. Further, the conformational difference between Boc5 and WB4-24, two closed related compounds, provides a structural framework for fine-tuning of pharmacological efficacy in the development of future small-molecule therapeutics targeting GLP-1R.

Identification and mechanism of G protein-biased ligands for chemokine receptor CCR1.

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-Gi complex 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 ß-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.

Orthosteric and allosteric modulation of human HCAR2 signaling complex.

Hydroxycarboxylic acids are crucial metabolic intermediates involved in various physiological and pathological processes, some of which are recognized by specific hydroxycarboxylic acid receptors (HCARs). HCAR2 is one such receptor, activated by endogenous ß-hydroxybutyrate (3-HB) and butyrate, and is the target for Niacin. Interest in HCAR2 has been driven by its potential as a therapeutic target in cardiovascular and neuroinflammatory diseases. However, the limited understanding of how ligands bind to this receptor has hindered the development of alternative drugs able to avoid the common flushing side-effects associated with Niacin therapy. Here, we present three high-resolution structures of HCAR2-Gi1 complexes bound to four different ligands, one potent synthetic agonist (MK-6892) bound alone, and the two structures bound to the allosteric agonist compound 9n in conjunction with either the endogenous ligand 3-HB or niacin. These structures coupled with our functional and computational analyses further our understanding of ligand recognition, allosteric modulation, and activation of HCAR2 and pave the way for the development of high-efficiency drugs with reduced side-effects.

Unsaturated bond recognition leads to biased signal in a fatty acid receptor.

Individual free fatty acids (FAs) play important roles in metabolic homeostasis, many through engagement with more than 40G protein-coupled receptors. Searching for receptors to sense beneficial omega-3 FAs of fish oil enabled the identification of GPR120, which is involved in a spectrum of metabolic diseases. Here, we report six cryo-electron microscopy structures of GPR120 in complex with FA hormones or TUG891 and Gi or Giq trimers. Aromatic residues inside the GPR120 ligand pocket were responsible for recognizing different double-bond positions of these FAs and connect ligand recognition to distinct effector coupling. We also investigated synthetic ligand selectivity and the structural basis of missense single-nucleotide polymorphisms. We reveal how GPR120 differentiates rigid double bonds and flexible single bonds. The knowledge gleaned here may facilitate rational drug design targeting to GPR120.

Structural insights into human brain-gut peptide cholecystokinin receptors.

The intestinal hormone and neuromodulator cholecystokinin (CCK) receptors CCK1R and CCK2R act as a signaling hub in brain-gut axis, mediating digestion, emotion, and memory regulation. CCK receptors exhibit distinct preferences for ligands in different posttranslational modification (PTM) states. CCK1R couples to Gs and Gq, whereas CCK2R primarily couples to Gq. Here we report the cryo-electron microscopy (cryo-EM) structures of CCK1R-Gs signaling complexes liganded either by sulfated cholecystokinin octapeptide (CCK-8) or a CCK1R-selective small-molecule SR146131, and CCK2R-Gq complexes stabilized by either sulfated CCK-8 or a CCK2R-selective ligand gastrin-17. Our structures reveal a location-conserved yet charge-distinct pocket discriminating the effects of ligand PTM states on receptor subtype preference, the unique pocket topology underlying selectivity of SR146131 and gastrin-17, the conformational changes in receptor activation, and key residues contributing to G protein subtype specificity, providing multiple structural templates for drug design targeting the brain-gut axis.

Structural insights into multiplexed pharmacological actions of tirzepatide and peptide 20 at the GIP, GLP-1 or glucagon receptors.

Glucose homeostasis, regulated by glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1) and glucagon (GCG) is critical to human health. Several multi-targeting agonists at GIPR, GLP-1R or GCGR, developed to maximize metabolic benefits with reduced side-effects, are in clinical trials to treat type 2 diabetes and obesity. To elucidate the molecular mechanisms by which tirzepatide, a GIPR/GLP-1R dual agonist, and peptide 20, a GIPR/GLP-1R/GCGR triagonist, manifest their multiplexed pharmacological actions over monoagonists such as semaglutide, we determine cryo-electron microscopy structures of tirzepatide-bound GIPR and GLP-1R as well as peptide 20-bound GIPR, GLP-1R and GCGR. The structures reveal both common and unique features for the dual and triple agonism by illustrating key interactions of clinical relevance at the near-atomic level. Retention of glucagon function is required to achieve such an advantage over GLP-1 monotherapy. Our findings provide valuable insights into the structural basis of functional versatility of tirzepatide and peptide 20.

Molecular basis for allosteric agonism and G protein subtype selectivity of galanin receptors.

Peptide hormones and neuropeptides are complex signaling molecules that predominately function through G protein-coupled receptors (GPCRs). Two unanswered questions remaining in the field of peptide-GPCR signaling systems pertain to the basis for the diverse binding modes of peptide ligands and the specificity of G protein coupling. Here, we report the structures of a neuropeptide, galanin, bound to its receptors, GAL1R and GAL2R, in complex with their primary G protein subtypes Gi and Gq, respectively. The structures reveal a unique binding pose of galanin, which almost 'lays flat' on the top of the receptor transmembrane domain pocket in an α-helical conformation, and acts as an 'allosteric-like' agonist via a distinct signal transduction cascade. The structures also uncover the important features of intracellular loop 2 (ICL2) that mediate specific interactions with Gq, thus determining the selective coupling of Gq to GAL2R. ICL2 replacement in Gi-coupled GAL1R, µOR, 5-HT1AR, and Gs-coupled ß2AR and D1R with that of GAL2R promotes Gq coupling of these receptors, highlighting the dominant roles of ICL2 in Gq selectivity. Together our results provide insights into peptide ligand recognition and allosteric activation of galanin receptors and uncover a general structural element for Gq coupling selectivity.

Molecular insights into ligand recognition and activation of chemokine receptors CCR2 and CCR3.

Chemokine receptors are a family of G-protein-coupled receptors with key roles in leukocyte migration and inflammatory responses. Here, we present cryo-electron microscopy structures of two human CC chemokine receptor-G-protein complexes: CCR2 bound to its endogenous ligand CCL2, and CCR3 in the apo state. The structure of the CCL2-CCR2-G-protein complex reveals that CCL2 inserts deeply into the extracellular half of the transmembrane domain, and forms substantial interactions with the receptor through the most N-terminal glutamine. Extensive hydrophobic and polar interactions are present between both two chemokine receptors and the Gα-protein, contributing to the constitutive activity of these receptors. Notably, complemented with functional experiments, the interactions around intracellular loop 2 of the receptors are found to be conserved and play a more critical role in G-protein activation than those around intracellular loop 3. Together, our findings provide structural insights into chemokine recognition and receptor activation, shedding lights on drug design targeting chemokine receptors.

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To get an optimal mode of irrigation and nitrogen supply for table grape production in North China, a pot experiment was conducted to investigate the effects of different irrigation modes and N application rates on dry matter accumulation and distribution, yield, water use efficiency, and nitrogen use efficiency of table grape. The irrigation modes included conventional drip irrigation (CDI, with sufficient irrigation), alternate partial root-zone drip irrigation (ADI, with 50% amount of the irrigation water of CDI) and fixed partial root-zone drip irrigation (FDI, with 50% amount of the irrigation water of CDI). The nitrogen application rates were set at 0.4 (N1), 0.8 (N2) and 1.2 (N3) g·kg-1 dry soil. The results showed that compared with CDI, ADI and FDI reduced new shoot pruning amount by 34.8% and 11.2%, respectively. New shoot pruning amount increased with increasing N application rates, being highest under CDIN3. Dry matter accumulation of ADI was the highest, being 5.1% and 12.8% higher than CDI and FDI. Dry matter accumulation was higher under N2 and N3 than N1. Compared with CDI and FDI, leaf to fruit ratio reduced but harvest index significantly increased in ADI, while those variables showed no significant difference among diffe-rent N application rates. The ratio of pruning amount to the biomass accumulated in the current year in ADIN2 was the lowest among the treatments. Compared with CDI and FDI, ADI increased grape fruit yield by 6.0% and 10.4%, respectively. Fruit yield was enhanced with increasing nitrogen application rates under the same irrigation condition, with the highest yield under the ADIN2 and ADIN3. Water use efficiency (WUE) increased significantly in ADI compared with CDI and FDI, with the highest value being observed in ADI coupled with N2 or N3. Nitrogen use efficiency (NUE) showed a trend of ADI＞CDI＞FDI. In addition, NUE decreased with increasing nitrogen supply level across the irrigation modes. In conclusion, ADIN2 could reduce the redundant growth of grape tree, promote the transfer of dry matter to fruit, which increased yield and use efficiency of both water and nitrogen, which is a suitable coupling water and nitrogen supply mode for grape production in northern China.

Molecular insights into ago-allosteric modulation of the human glucagon-like peptide-1 receptor.

The glucagon-like peptide-1 (GLP-1) receptor is a validated drug target for metabolic disorders. Ago-allosteric modulators are capable of acting both as agonists on their own and as efficacy enhancers of orthosteric ligands. However, the molecular details of ago-allosterism remain elusive. Here, we report three cryo-electron microscopy structures of GLP-1R bound to (i) compound 2 (an ago-allosteric modulator); (ii) compound 2 and GLP-1; and (iii) compound 2 and LY3502970 (a small molecule agonist), all in complex with heterotrimeric Gs. The structures reveal that compound 2 is covalently bonded to C347 at the cytoplasmic end of TM6 and triggers its outward movement in cooperation with the ECD whose N terminus penetrates into the GLP-1 binding site. This allows compound 2 to execute positive allosteric modulation through enhancement of both agonist binding and G protein coupling. Our findings offer insights into the structural basis of ago-allosterism at GLP-1R and may aid the design of better therapeutics.

Structural insights into ligand recognition and activation of the melanocortin-4 receptor.

Melanocortin-4 receptor (MC4R) plays a central role in the regulation of energy homeostasis. Its high sequence similarity to other MC receptor family members, low agonist selectivity and the lack of structural information concerning MC4R-specific activation have hampered the development of MC4R-seletive therapeutics to treat obesity. Here, we report four high-resolution structures of full-length MC4R in complex with the heterotrimeric Gs protein stimulated by the endogenous peptide ligand α-MSH, FDA-approved drugs afamelanotide (Scenesse™) and bremelanotide (Vyleesi™), and a selective small-molecule ligand THIQ, respectively. Together with pharmacological studies, our results reveal the conserved binding mode of peptidic agonists, the distinctive molecular details of small-molecule agonist recognition underlying receptor subtype selectivity, and a distinct activation mechanism for MC4R, thereby offering new insights into G protein coupling. Our work may facilitate the discovery of selective therapeutic agents targeting MC4R.

Cryo-EM structures of inactive and active GABAB receptor.

Metabotropic GABAB G protein-coupled receptor functions as a mandatory heterodimer of GB1 and GB2 subunits and mediates inhibitory neurotransmission in the central nervous system. Each subunit is composed of the extracellular Venus flytrap (VFT) domain and transmembrane (TM) domain. Here we present cryo-EM structures of full-length human heterodimeric GABAB receptor in the antagonist-bound inactive state and in the active state complexed with an agonist and a positive allosteric modulator in the presence of Gi1 protein at a resolution range of 2.8-3.0 Å. Our structures reveal that agonist binding stabilizes the closure of GB1 VFT, which in turn triggers a rearrangement of TM interfaces between the two subunits from TM3-TM5/TM3-TM5 in the inactive state to TM6/TM6 in the active state and finally induces the opening of intracellular loop 3 and synergistic shifting of TM3, 4 and 5 helices in GB2 TM domain to accommodate the α5-helix of Gi1. We also observed that the positive allosteric modulator anchors at the dimeric interface of TM domains. These results provide a structural framework for understanding class C GPCR activation and a rational template for allosteric modulator design targeting the dimeric interface of GABAB receptor.

A unique hormonal recognition feature of the human glucagon-like peptide-2 receptor.

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 Gs heterotrimer. 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.

Dysregulated renin-angiotensin system contributes to acute lung injury caused by hind-limb ischemia-reperfusion in mice.

The mechanism of acute lung injury (ALI) following limb ischemia-reperfusion (LIR) is not yet clear. We speculate that the unbalanced expression of angiotensin-converting enzymes (ACE and ACE2) and angiotensins [Ang II and Ang-(1-7)] in the renin-angiotensin system (RAS) is a major cause of ALI. To prove this hypothesis, pathological changes, lung edema, and permeability of wild-type mice at different time points within 12 h of reperfusion after 2 h of hind-limb ischemia were first detected by morphological method, measurements of wet-to-dry weight ratio, and bronchoalveolar lavage fluid. Meanwhile, the changes of lung ACE/ACE2 mRNA and protein expression were surveyed by the methods of real-time reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemistry. Angiotensin II/Ang-(1-7) levels in the blood serum and lung tissue were measured by enzyme-linked immunosorbent assay. Then the effects of ACE2 gene insertion and deletion on the previously mentioned parameters were investigated in the mice being exposed to hind-limb 2-h ischemia and 4-h reperfusion. The results revealed that lung injuries in the wild-type mice were gradually aggravated, and the expression of ACE in lung tissue was progressively increased, whereas that of ACE2 decreased within 12 h after LIR. Unexpectedly, both Ang II and Ang-(1-7) in the lung tissue were obviously increased after LIR, showing Ang-(1-7) higher than Ang II in the early stage of reperfusion but lower than Ang II at the late stage of reperfusion. Unlike local Ang II/Ang-(1-7) changes, circulating Ang-(1-7) became greatly descending, and Ang II was markedly ascending from the start of reperfusion, corresponding to local ACE/ACE2 unbalanced expression. ACE2 transgenosis improved the imbalance of ACE/ACE2 and Ang II/Ang-(1-7) expression and alleviated lung injuries, whereas ACE2 knockout further aggravated the imbalance of ACE/ACE2 and Ang II/Ang-(1-7) expression and made lung injuries more serious in the post-LIR mice. The results indicate that the dysregulation of local and circulating RAS with increased expression of ACE/Ang II and decreased expression of ACE2/Ang-(1-7) contribute to ALI caused by LIR in mice. Maintaining RAS homeostasis through upregulating ACE2 expression may lessen lung injury, which provides a new idea for the treatment of posttraumatic ALI.