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 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 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.

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 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.

Drought Stress Influences the Growth and Physiological Characteristics of Solanum rostratum Dunal Seedlings From Different Geographical Populations in China.

Extensive studies have shown that the success of invasive plants in large environmental gradients can be partly attributed to related factors, including phenotypic plasticity and rapid evolution. To enhance their ability to compete and invade, invasive plants often show higher morphological and physiological plasticity to adapt to different habitat conditions. In the past two decades, invasive species have expanded to some new habitats in North and Northwest China, including arid oasis agricultural zones, which are disturbed by human activities, and the ecosystem itself is very fragile. To evaluate the ecological adaptability of invasive plants widely distributed in North and Northwest China, we studied the physiological response and tolerance mechanism of different geographical populations of Solanum rostratum Dunal to different drought-stress gradients in extremely arid regions (Xinjiang population) and semi-arid regions (Inner Mongolia population). The results showed that with the aggravation of drought stress, S. rostratum from different geographical populations adopted different physiological mechanisms to drought stress. Xinjiang population was mostly affected by root/shoot ratio and chlorophyll fluorescence characteristics, showing higher plasticity in the net and total photosynthetic rates, while the Inner Mongolia population mainly relied on the accumulation of osmotic adjustment substances, higher leaf dry matter content, and increased malondialdehyde to cope with drought stress. Based on these results, we concluded that the physiological responses of S. rostratum invading different habitats in northern China to drought stress were significantly different. The drought resistance of the Xinjiang population was higher than that of the Inner Mongolia population. In general, S. rostratum can be widely adapted to both harsh and mild habitats through phenotypic plasticity, threatening agricultural production and ecological environment security in northern China.

Cryo-EM structure of the human histamine H1 receptor/Gq complex.

Histamine receptors play important roles in various pathophysiological conditions and are effective targets for anti-allergy treatment, however the mechanism of receptor activation remain elusive. Here, we present the cryo-electron microscopy (cryo-EM) structure of the human H1R in complex with a Gq protein in an active conformation via a NanoBiT tethering strategy. The structure reveals that histamine activates receptor via interacting with the key residues of both transmembrane domain 3 (TM3) and TM6 to squash the binding pocket on the extracellular side and to open the cavity on the intracellular side for Gq engagement in a model of "squash to activate and expand to deactivate". The structure also reveals features for Gq coupling, including the interaction between intracellular loop 2 (ICL2) and the αN-ß junction of Gq/11 protein. The detailed analysis of our structure will provide a framework for understanding G-protein coupling selectivity and clues for designing novel antihistamines.

Leaf Physiological Responses of Three Psammophytes to Combined Effects of Warming and Precipitation Reduction in Horqin Sandy Land, Northeast China.

The decreasing precipitation with global climate warming is the main climatic condition in some sandy grassland ecosystems. The understanding of physiological responses of psammophytes in relation to warming and precipitation is a possible way to estimate the response of plant community stability to climate change. We selected Lespedeza davurica, Artemisia scoparia, and Cleistogenes squarrosa in sandy grassland to examine the effect of a combination of climate warming and decreasing precipitation on relative water content (RWC), chlorophyll, proline, and antioxidant enzyme activities. We found that all experimental treatments have influenced RWC, chlorophyll, proline, and antioxidant enzyme activities of three psammophytes. L. davurica has the highest leaf RWC among the three psammophytes. With the intensification of precipitation reduction, the decreasing amplitude of chlorophyll from three psammophytes was L. davurica > C. squarrosa > A. scoparia. At the natural temperature, the malondialdehyde (MDA) content of the three psammophytes under severe drought treatment was much higher than other treatments, and their increasing degree was as follows: A. scoparia > C. squarrosa > L. davurica. At the same precipitation gradient, the proline of three psammophytes under warming was higher than the natural temperature. The differences in superoxide dismutase (SOD) among the three psammophytes were A. scoparia > L. davurica > C. squarrosa. Moreover, at natural temperature, more than 40% of precipitation reduction was most significant. Regardless of warming or not, the catalase (CAT) activity of A. scoparia under reduced precipitation treatments was higher than natural temperature, while the response of L. davurica was opposite. Correlation analyses evidenced that warming (T) was significant in L. davurica and precipitation (W) was significant in A. scoparia and C. squarrosa according to the Monte-Carlo permutation test (p = 0.002, 0.004, and 0.004). The study is important in predicting how local plants will respond to future climate change and assessing the possible effects of climate change on sandy grassland ecosystems.

A complex structure of arrestin-2 bound to a G protein-coupled receptor.

Arrestins comprise a family of signal regulators of G-protein-coupled receptors (GPCRs), which include arrestins 1 to 4. While arrestins 1 and 4 are visual arrestins dedicated to rhodopsin, arrestins 2 and 3 (Arr2 and Arr3) are ß-arrestins known to regulate many nonvisual GPCRs. The dynamic and promiscuous coupling of Arr2 to nonvisual GPCRs has posed technical challenges to tackle the basis of arrestin binding to GPCRs. Here we report the structure of Arr2 in complex with neurotensin receptor 1 (NTSR1), which reveals an overall assembly that is strikingly different from the visual arrestin-rhodopsin complex by a 90° rotation of Arr2 relative to the receptor. In this new configuration, intracellular loop 3 (ICL3) and transmembrane helix 6 (TM6) of the receptor are oriented toward the N-terminal domain of the arrestin, making it possible for GPCRs that lack the C-terminal tail to couple Arr2 through their ICL3. Molecular dynamics simulation and crosslinking data further support the assembly of the Arr2‒NTSR1 complex. Sequence analysis and homology modeling suggest that the Arr2‒NTSR1 complex structure may provide an alternative template for modeling arrestin-GPCR interactions.

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 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.

Ginkgolic acid, a sumoylation inhibitor, promotes adipocyte commitment but suppresses adipocyte terminal differentiation of mouse bone marrow stromal cells.

Sumoylation is a post-translational modification process having an important influence in mesenchymal stem cell (MSC) differentiation. Thus, sumoylation-modulating chemicals might be used to control MSC differentiation for skeletal tissue engineering. In this work, we studied how the differentiation of mouse bone marrow stromal cells (mBMSCs) is affected by ginkgolic acid (GA), a potent sumoylation inhibitor also reported to inhibit histone acetylation transferase (HAT). Our results show that GA promoted the differentiation of mBMSCs into adipocytes when cultured in osteogenic medium. Moreover, mBMSCs pre-treated with GA showed enhanced pre-adipogenic gene expression and were more efficiently differentiated into adipocytes when subsequently cultured in the adipogenic medium. However, when GA was added at a later stage of adipogenesis, adipocyte maturation was markedly inhibited, with a dramatic down-regulation of multiple lipogenesis genes. Moreover, we found that the effects of garcinol, a HAT inhibitor, differed from those of GA in regulating adipocyte commitment and adipocyte maturation of mBMSCs, implying that the GA function in adipogenesis is likely through its activity as a sumoylation inhibitor, not as a HAT inhibitor. Overall, our studies revealed an unprecedented role of GA in MSC differentiation and provide new mechanistic insights into the use of GA in clinical applications.