Identification of Phosphorylation Codes for Arrestin Recruitment by G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) mediate diverse signaling in part through interaction with arrestins, whose binding promotes receptor internalization and signaling through G protein-independent pathways. High-affinity arrestin binding requires receptor phosphorylation, often at the receptor's C-terminal tail. Here, we report an X-ray free electron laser (XFEL) crystal structure of the rhodopsin-arrestin complex, in which the phosphorylated C terminus of rhodopsin forms an extended intermolecular ß sheet with the N-terminal ß strands of arrestin. Phosphorylation was detected at rhodopsin C-terminal tail residues T336 and S338. These two phospho-residues, together with E341, form an extensive network of electrostatic interactions with three positively charged pockets in arrestin in a mode that resembles binding of the phosphorylated vasopressin-2 receptor tail to ß-arrestin-1. Based on these observations, we derived and validated a set of phosphorylation codes that serve as a common mechanism for phosphorylation-dependent recruitment of arrestins by GPCRs.

Structural insights into adhesion GPCR ADGRL3 activation and Gq, Gs, Gi, and G12 coupling.

Adhesion G-protein-coupled receptors (aGPCRs) play key roles in a diversity of physiologies. A hallmark of aGPCR activation is the removal of the inhibitory GAIN domain and the dipping of the cleaved stalk peptide into the ligand-binding pocket of receptors; however, the detailed mechanism remains obscure. Here, we present cryoelectron microscopy (cryo-EM) structures of ADGRL3 in complex with Gq, Gs, Gi, and G12. The structures reveal unique ligand-engaging mode, distinctive activation conformation, and key mechanisms of aGPCR activation. The structures also reveal the uncharted structural information of GPCR/G12 coupling. A comparison of Gq, Gs, Gi, and G12 engagements with ADGRL3 reveals the key determinant of G-protein coupling on the far end of αH5 of Gα. A detailed analysis of the engagements allows us to design mutations that specifically enhance one pathway over others. Taken together, our study lays the groundwork for understanding aGPCR activation and G-protein-coupling selectivity.

Structural basis of sphingosine-1-phosphate receptor 1 activation and biased agonism.

Sphingosine-1-phosphate receptor 1 (S1PR1) is a master regulator of lymphocyte egress from the lymph node and an established drug target for multiple sclerosis (MS). Mechanistically, therapeutic S1PR1 modulators activate the receptor yet induce sustained internalization through a potent association with ß-arrestin. However, a structural basis of biased agonism remains elusive. Here, we report the cryo-electron microscopy (cryo-EM) structures of Gi-bound S1PR1 in complex with S1P, fingolimod-phosphate (FTY720-P) and siponimod (BAF312). In combination with functional assays and molecular dynamics (MD) studies, we reveal that the ß-arrestin-biased ligands direct a distinct activation path in S1PR1 through the extensive interplay between the PIF and the NPxxY motifs. Specifically, the intermediate flipping of W2696.48 and the retained interaction between F2656.44 and N3077.49 are the key features of the ß-arrestin bias. We further identify ligand-receptor interactions accounting for the S1PR subtype specificity of BAF312. These structural insights provide a rational basis for designing novel signaling-biased S1PR modulators.

Crystal structure of the Frizzled 4 receptor in a ligand-free state.

Frizzled receptors (FZDs) are class-F G-protein-coupled receptors (GPCRs) that function in Wnt signalling and are essential for developing and adult organisms1,2. As central mediators in this complex signalling pathway, FZDs serve as gatekeeping proteins both for drug intervention and for the development of probes in basic and in therapeutic research. Here we present an atomic-resolution structure of the human Frizzled 4 receptor (FZD4) transmembrane domain in the absence of a bound ligand. The structure reveals an unusual transmembrane architecture in which helix VI is short and tightly packed, and is distinct from all other GPCR structures reported so far. Within this unique transmembrane fold is an extremely narrow and highly hydrophilic pocket that is not amenable to the binding of traditional GPCR ligands. We show that such a pocket is conserved across all FZDs, which may explain the long-standing difficulties in the development of ligands for these receptors. Molecular dynamics simulations on the microsecond timescale and mutational analysis uncovered two coupled, dynamic kinks located at helix VII that are involved in FZD4 activation. The stability of the structure in its ligand-free form, an unfavourable pocket for ligand binding and the two unusual kinks on helix VII suggest that FZDs may have evolved a novel ligand-recognition and activation mechanism that is distinct from that of other GPCRs.

Structural basis for corepressor assembly by the orphan nuclear receptor TLX.

The orphan nuclear receptor TLX regulates neural stem cell self-renewal in the adult brain and functions primarily as a transcription repressor through recruitment of Atrophin corepressors, which bind to TLX via a conserved peptide motif termed the Atro box. Here we report crystal structures of the human and insect TLX ligand-binding domain in complex with Atro box peptides. In these structures, TLX adopts an autorepressed conformation in which its helix H12 occupies the coactivator-binding groove. Unexpectedly, H12 in this autorepressed conformation forms a novel binding pocket with residues from helix H3 that accommodates a short helix formed by the conserved ALXXLXXY motif of the Atro box. Mutations that weaken the TLX-Atrophin interaction compromise the repressive activity of TLX, demonstrating that this interaction is required for Atrophin to confer repressor activity to TLX. Moreover, the autorepressed conformation is conserved in the repressor class of orphan nuclear receptors, and mutations of corresponding residues in other members of this class of receptors diminish their repressor activities. Together, our results establish the functional conservation of the autorepressed conformation and define a key sequence motif in the Atro box that is essential for TLX-mediated repression.

Development of highly potent glucocorticoids for steroid-resistant severe asthma.

Clinical application of inhaled glucocorticoids (GCs) has been hampered in the case of steroid-resistant severe asthma. To overcome this limitation, we have developed a series of highly potent GCs, including VSGC12, VSG158, and VSG159 based on the structural insight into the glucocorticoid receptor (GR). Particularly, VSG158 exhibits a maximal repression of lung inflammation and is 10 times more potent than the currently most potent clinical GC, Fluticasone Furoate (FF), in a murine model of asthma. More importantly, VSG158 displays a unique property to reduce neutrophilic inflammation in a steroid-resistant airway inflammation model, which is refractory to clinically available GCs, including dexamethasone and FF. VSG158 and VSG159 are able to deliver effective treatments with reduced off-target and side effects. In addition, these GCs also display pharmacokinetic properties that are suitable for the inhalation delivery method for asthma treatment. Taken together, the excellent therapeutic and side-effect profile of these highly potent GCs holds promise for treating steroid-resistant severe asthma.

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.

DWARF 53 acts as a repressor of strigolactone signalling in rice.

Strigolactones (SLs) are a group of newly identified plant hormones that control plant shoot branching. SL signalling requires the hormone-dependent interaction of DWARF 14 (D14), a probable candidate SL receptor, with DWARF 3 (D3), an F-box component of the Skp-Cullin-F-box (SCF) E3 ubiquitin ligase complex. Here we report the characterization of a dominant SL-insensitive rice (Oryza sativa) mutant dwarf 53 (d53) and the cloning of D53, which encodes a substrate of the SCF(D3) ubiquitination complex and functions as a repressor of SL signalling. Treatments with GR24, a synthetic SL analogue, cause D53 degradation via the proteasome in a manner that requires D14 and the SCF(D3) ubiquitin ligase, whereas the dominant form of D53 is resistant to SL-mediated degradation. Moreover, D53 can interact with transcriptional co-repressors known as TOPLESS-RELATED PROTEINS. Our results suggest a model of SL signalling that involves SL-dependent degradation of the D53 repressor mediated by the D14-D3 complex.

Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors.

The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical ß2-adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation.

Structural insights into gene repression by the orphan nuclear receptor SHP.

Small heterodimer partner (SHP) is an orphan nuclear receptor that functions as a transcriptional repressor to regulate bile acid and cholesterol homeostasis. Although the precise mechanism whereby SHP represses transcription is not known, E1A-like inhibitor of differentiation (EID1) was isolated as a SHP-interacting protein and implicated in SHP repression. Here we present the crystal structure of SHP in complex with EID1, which reveals an unexpected EID1-binding site on SHP. Unlike the classical cofactor-binding site near the C-terminal helix H12, the EID1-binding site is located at the N terminus of the receptor, where EID1 mimics helix H1 of the nuclear receptor ligand-binding domain. The residues composing the SHP-EID1 interface are highly conserved. Their mutation diminishes SHP-EID1 interactions and affects SHP repressor activity. Together, these results provide important structural insights into SHP cofactor recruitment and repressor function and reveal a conserved protein interface that is likely to have broad implications for transcriptional repression by orphan nuclear receptors.

Disruption of Ttll5/stamp gene (tubulin tyrosine ligase-like protein 5/SRC-1 and TIF2-associated modulatory protein gene) in male mice causes sperm malformation and infertility.

TTLL5/STAMP (tubulin tyrosine ligase-like family member 5) has multiple activities in cells. TTLL5 is one of 13 TTLLs, has polyglutamylation activity, augments the activity of p160 coactivators (SRC-1 and TIF2) in glucocorticoid receptor-regulated gene induction and repression, and displays steroid-independent growth activity with several cell types. To examine TTLL5/STAMP functions in whole animals, mice were prepared with an internal deletion that eliminated several activities of the Stamp gene. This mutation causes both reduced levels of STAMP mRNA and C-terminal truncation of STAMP protein. Homozygous targeted mutant (Stamp(tm/tm)) mice appear normal except for marked decreases in male fertility associated with defects in progressive sperm motility. Abnormal axonemal structures with loss of tubulin doublets occur in most Stamp(tm/tm) sperm tails in conjunction with substantial reduction in α-tubulin polyglutamylation, which closely correlates with the reduction in mutant STAMP mRNA. The axonemes in other structures appear unaffected. There is no obvious change in the organs for sperm development of WT versus Stamp(tm/tm) males despite the levels of WT STAMP mRNA in testes being 20-fold higher than in any other organ examined. This defect in male fertility is unrelated to other Ttll genes or 24 genes previously identified as important for sperm function. Thus, STAMP appears to participate in a unique, tissue-selective TTLL-mediated pathway for α-tubulin polyglutamylation that is required for sperm maturation and motility and may be relevant for male fertility.

Biodiversity drives ecosystem multifunctionality in sandy grasslands?

Plant communities and soil microbiomes play a crucial role in regulating ecosystem multifunctionality (EMF). However, whether and how aboveground plant diversity, belowground soil microbial diversity and interactions with environmental factors affect EMF in sandy grasslands under climate change conditions is unclear. Here, we selected 15 typical grassland communities from the Horqin sandy grassland along temperature and precipitation gradients, using the mean annual temperature (AMT), mean annual precipitation (AP), soil temperature (ST), soil water content (SW) and pH as abiotic factors, and plant diversity (PD) and soil microbial diversity (SD) as biodiversity indicators. The effects of biodiversity and abiotic factors on individual ecosystem functions and EMF were studied. We found that PD and its components, plant species richness (SR), species diversity (PR) and genetic diversity (GD), had significant effects on aboveground biomass (AGB) and major factors involved in ecosystem nitrogen cycling (plant leaf nitrogen content (PLN) and soil total nitrogen content (STN)) (P < 0.05). Soil fungal diversity (FR) has a greater impact on ecosystem function than soil bacteria (BR) and archaea (ABR) in sandy grasslands and mainly promotes the accumulation of soil microbial carbon and nitrogen (MBC, MBN) (P < 0.05), STC and STN (P < 0.01). PD and two types of SD (FR and ABR) significantly regulated EMF (P < 0.01). Among the abiotic factors, soil pH and SW regulated EMF (P < 0.05), and SW and ST directly drove EMF (P < 0.05). PD drove EMF significantly and indirectly (positively) through soil pH and ST (P < 0.001), while SD drove EMF weakly and indirectly (negatively) through AP and PD (P > 0.05). PD was a stronger driving force on EMF than SD. These results improve our understanding of the drivers of multifunctionality in sandy grassland ecosystems.

Structural basis of ligand recognition and design of antihistamines targeting histamine H4 receptor.

The histamine H4 receptor (H4R) plays key role in immune cell function and is a highly valued target for treating allergic and inflammatory diseases. However, structural information of H4R remains elusive. Here, we report four cryo-EM structures of H4R/Gi complexes, with either histamine or synthetic agonists clobenpropit, VUF6884 and clozapine bound. Combined with mutagenesis, ligand binding and functional assays, the structural data reveal a distinct ligand binding mode where D943.32 and a π-π network determine the orientation of the positively charged group of ligands, while E1825.46, located at the opposite end of the ligand binding pocket, plays a key role in regulating receptor activity. The structural insight into H4R ligand binding allows us to identify mutants at E1825.46 for which the agonist clobenpropit acts as an inverse agonist and to correctly predict inverse agonism of a closely related analog with nanomolar potency. Together with the findings regarding receptor activation and Gi engagement, we establish a framework for understanding H4R signaling and provide a rational basis for designing novel antihistamines targeting H4R.

Identification of oleic acid as an endogenous ligand of GPR3.

Although GPR3 plays pivotal roles in both the nervous system and metabolic processes, such as cold-induced thermogenesis, its endogenous ligand remains elusive. Here, by combining structural approach (including cryo-electron microscopy), mass spectrometry analysis, and functional studies, we identify oleic acid (OA) as an endogenous ligand of GPR3. Our study reveals a hydrophobic tunnel within GPR3 that connects the extracellular side of the receptor to the middle of plasma membrane, enabling fatty acids to readily engage the receptor. Functional studies demonstrate that OA triggers downstream Gs signaling, whereas lysophospholipids fail to activate the receptor. Moreover, our research reveals that cold stimulation induces the secretion of OA in mice, subsequently activating Gs/cAMP/PKA signaling in brown adipose tissue. Notably, brown adipose tissues from Gpr3 knockout mice do not respond to OA during cold stimulation, reinforcing the significance of GPR3 in this process. Finally, we propose a "born to be activated and cold to enhance" model for GPR3 activation. Our study provides a starting framework for the understanding of GPR3 signaling in cold-stimulated thermogenesis.

Structural basis of CD97 activation and G-protein coupling.

CD97 (ADGRE5) is an adhesion G protein-coupled receptor (aGPCR) which plays crucial roles in immune system and cancer. However, the mechanism of CD97 activation and the determinant of G13 coupling selectivity remain unknown. Here, we present the cryo-electron microscopy structures of human CD97 in complex with G13, Gq, and Gs. Our structures reveal the stalk peptide recognition mode of CD97, adding missing information of the current tethered-peptide activation model of aGPCRs. For instance, a revised "FXφφφ" motif and a framework of conserved aromatic residues in the ligand-binding pocket. Importantly, structural comparisons of G13, Gq, and Gs engagements of CD97 reveal key determinants of G13 coupling selectivity, where a deep insertion of the α helix 5 and a closer contact with the transmembrane helix 6, 5, and 3 dictate coupling preferences. Taken together, our structural study of CD97 provides a framework for understanding CD97 signaling and the G13 coupling selectivity.

Structural basis of lysophosphatidylserine receptor GPR174 ligand recognition and activation.

Lysophosphatidylserine (LysoPS) is a lipid mediator that induces multiple cellular responses through binding to GPR174. Here, we present the cryo-electron microscopy (cryo-EM) structure of LysoPS-bound human GPR174 in complex with Gs protein. The structure reveals a ligand recognition mode, including the negatively charged head group of LysoPS forms extensive polar interactions with surrounding key residues of the ligand binding pocket, and the L-serine moiety buries deeply into a positive charged cavity in the pocket. In addition, the structure unveils a partially open pocket on transmembrane domain helix (TM) 4 and 5 for a lateral entry of ligand. Finally, the structure reveals a Gs engaging mode featured by a deep insertion of a helix 5 (αH5) and extensive polar interactions between receptor and αH5. Taken together, the information revealed by our structural study provides a framework for understanding LysoPS signaling and a rational basis for designing LysoPS receptor-targeting drugs.

Structural basis of leukotriene B4 receptor 1 activation.

Leukotriene B4 receptor 1 (BLT1) plays crucial roles in the acute inflammatory responses and is a valuable target for anti-inflammation treatment, however, the mechanism by which leukotriene B4 (LTB4) activates receptor remains unclear. Here, we report the cryo-electron microscopy (cryo-EM) structure of the LTB4 -bound human BLT1 in complex with a Gi protein in an active conformation at resolution of 2.91 Å. In combination of molecule dynamics (MD) simulation, docking and site-directed mutagenesis, our structure reveals that a hydrogen-bond network of water molecules and key polar residues is the key molecular determinant for LTB4 binding. We also find that the displacement of residues M1013.36 and I2717.39 to the center of receptor, which unlock the ion lock of the lower part of pocket, is the key mechanism of receptor activation. In addition, we reveal a binding site of phosphatidylinositol (PI) and discover that the widely open ligand binding pocket may contribute the lack of specificity and efficacy for current BLT1-targeting drug design. Taken together, our structural analysis provides a scaffold for understanding BLT1 activation and a rational basis for designing anti-leukotriene drugs.

Structural basis of adhesion GPCR GPR110 activation by stalk peptide and G-proteins coupling.

Adhesion G protein-coupled receptors (aGPCRs) are keys of many physiological events and attractive targets for various diseases. aGPCRs are also known to be capable of self-activation via an autoproteolysis process that removes the inhibitory GAIN domain on the extracellular side of receptor and releases a stalk peptide to bind and activate the transmembrane side of receptor. However, the detailed mechanism of aGPCR activation remains elusive. Here, we report the cryo-electron microscopy structures of GPR110 (ADGRF1), a member of aGPCR, in complex with Gq, Gs, Gi, G12 and G13. The structures reveal distinctive ligand engaging model and activation conformations of GPR110. The structures also unveil the rarely explored GPCR/G12 and GPCR/G13 engagements. A comparison of Gq, Gs, Gi, G12 and G13 engagements with GPR110 reveals details of G-protein engagement, including a dividing point at the far end of the alpha helix 5 (αH5) of Gα subunit that separates Gq/Gs engagements from Gi/G12/G13 engagements. This is also where Gq/Gs bind the receptor through both hydrophobic and polar interaction, while Gi/G12/G13 engage receptor mainly through hydrophobic interaction. We further provide physiological evidence of GPR110 activation via stalk peptide. Taken together, our study fills the missing information of GPCR/G-protein engagement and provides a framework for understanding aGPCR activation and GPR110 signaling.

Identification of SRC3/AIB1 as a preferred coactivator for hormone-activated androgen receptor.

Transcription activation by androgen receptor (AR), which depends on recruitment of coactivators, is required for the initiation and progression of prostate cancer, yet the mechanisms of how hormone-activated AR interacts with coactivators remain unclear. This is because AR, unlike any other nuclear receptor, prefers its own N-terminal FXXLF motif to the canonical LXXLL motifs of coactivators. Through biochemical and crystallographic studies, we identify that steroid receptor coactivator-3 (SRC3) (also named as amplified in breast cancer-1 or AIB1) interacts strongly with AR via synergistic binding of its first and third LXXLL motifs. Mutagenesis and functional studies confirm that SRC3 is a preferred coactivator for hormone-activated AR. Importantly, AR mutations found in prostate cancer patients correlate with their binding potency to SRC3, corroborating with the emerging role of SRC3 as a prostate cancer oncogene. These results provide a molecular mechanism for the selective utilization of SRC3 by hormone-activated AR, and they link the functional relationship between AR and SRC3 to the development and growth of prostate cancer.