Applications of cryo-EM in drug development for STING.

STING is a critical adaptor protein in the cGAS-mediated DNA-sensing innate immune pathway. Binding of the second messenger cGAMP generated by cGAS to STING induces the high-order oligomerization and activation of the STING dimer. STING is a promising target for diseases associated with the cGAS/STING pathway such as cancer and autoimmune diseases. Recent applications of cryo-EM to STING have led to exciting progress in the understanding of its regulatory mechanism. Cryo-EM structures of STING bound to either cGAMP mimetics or novel small molecule ligands not only revealed the action mechanisms of these ligands but also suggested new ways to modulate the activity of STING for therapeutic purposes. Some of these recent studies are highlighted here.

Activation of human STING by a molecular glue-like compound.

Stimulator of interferon genes (STING) is a dimeric transmembrane adapter protein that plays a key role in the human innate immune response to infection and has been therapeutically exploited for its antitumor activity. The activation of STING requires its high-order oligomerization, which could be induced by binding of the endogenous ligand, cGAMP, to the cytosolic ligand-binding domain. Here we report the discovery through functional screens of a class of compounds, named NVS-STGs, that activate human STING. Our cryo-EM structures show that NVS-STG2 induces the high-order oligomerization of human STING by binding to a pocket between the transmembrane domains of the neighboring STING dimers, effectively acting as a molecular glue. Our functional assays showed that NVS-STG2 could elicit potent STING-mediated immune responses in cells and antitumor activities in animal models.

The Activation Mechanism of the Insulin Receptor: A Structural Perspective.

The insulin receptor (IR) is a type II receptor tyrosine kinase that plays essential roles in metabolism, growth, and proliferation. Dysregulation of IR signaling is linked to many human diseases, such as diabetes and cancers. The resolution revolution in cryo-electron microscopy has led to the determination of several structures of IR with different numbers of bound insulin molecules in recent years, which have tremendously improved our understanding of how IR is activated by insulin. Here, we review the insulin-induced activation mechanism of IR, including (a) the detailed binding modes and functions of insulin at site 1 and site 2 and (b) the insulin-induced structural transitions that are required for IR activation. We highlight several other key aspects of the activation and regulation of IR signaling and discuss the remaining gaps in our understanding of the IR activation mechanism and potential avenues of future research.

Ex utero intrapartum therapy in infants with congenital diaphragmatic hernia: a propensity score matching analysis.

Objective: Previous studies have shown that ex utero intrapartum therapy (EXIT) is safe and feasible for newborns with congenital diaphragmatic hernia (CDH). This study reports our experience with EXIT in fetuses with CDH in an attempt to explore the efficacy of EXIT on the survival rate of this population. Methods: A retrospective analysis of the clinical data of 116 children with CDH was conducted. The children were assigned to EXIT and non-EXIT groups. Propensity score matching (PSM) toward clinical data was performed, and the clinical characteristics and outcomes were compared. Taking survival at discharge as the main outcome, logistic regression analysis was carried out to explore the efficacy of EXIT on survival. Results: During the study period, 30 of 116 children received EXIT. After PSM, the survival rates of the EXIT group and the non-EXIT group were 82.76% (24/29) and 48.28% (14/29), respectively (p=0.006). EXIT (OR=0.083, 95% CI=0.013to 0.525, p=0.008), liver herniation (OR=16.955, 95% CI=2.342 to 122.767, p=0.005), and gestational age at diagnosis (OR=0.662, 95% CI=0.497 to 0.881, p=0.005) were independent mortality-related risk factors of all children with CDH. Ninety-nine of 116 children underwent surgery. After PSM, the postoperative survival rates of the EXIT group and non-EXIT group were 84.6% (22/26) and 76.9% (20/26), respectively (p=0.754). Liver herniation (OR=10.451, 95% CI=1.641 to 66.544, p=0.013) and gestational age at diagnosis (OR=0.736, 95% CI=0.577 to 0.938, p=0.013) were independent mortality-related risk factors of children after surgery. Conclusion: EXIT can be performed safely for selected prenatally diagnosed CDH neonates with potentially better survival and does not cause more maternal complications compared with traditional cesarean section.

Structural basis for gating mechanism of the human sodium-potassium pump.

P2-type ATPase sodium-potassium pumps (Na+/K+-ATPases) are ion-transporting enzymes that use ATP to transport Na+ and K+ on opposite sides of the lipid bilayer against their electrochemical gradients to maintain ion concentration gradients across the membranes in all animal cells. Despite the available molecular architecture of the Na+/K+-ATPases, a complete molecular mechanism by which the Na+ and K+ ions access into and are released from the pump remains unknown. Here we report five cryo-electron microscopy (cryo-EM) structures of the human alpha3 Na+/K+-ATPase in its cytoplasmic side-open (E1), ATP-bound cytoplasmic side-open (E1•ATP), ADP-AlF4- trapped Na+-occluded (E1•P-ADP), BeF3- trapped exoplasmic side-open (E2P) and MgF42- trapped K+-occluded (E2•Pi) states. Our work reveals the atomically resolved structural detail of the cytoplasmic gating mechanism of the Na+/K+-ATPase.

Activation of the insulin receptor by an insulin mimetic peptide.

Insulin receptor (IR) signaling defects cause a variety of metabolic diseases including diabetes. Moreover, inherited mutations of the IR cause severe insulin resistance, leading to early morbidity and mortality with limited therapeutic options. A previously reported selective IR agonist without sequence homology to insulin, S597, activates IR and mimics insulin's action on glycemic control. To elucidate the mechanism of IR activation by S597, we determine cryo-EM structures of the mouse IR/S597 complex. Unlike the compact T-shaped active IR resulting from the binding of four insulins to two distinct sites, two S597 molecules induce and stabilize an extended T-shaped IR through the simultaneous binding to both the L1 domain of one protomer and the FnIII-1 domain of another. Importantly, S597 fully activates IR mutants that disrupt insulin binding or destabilize the insulin-induced compact T-shape, thus eliciting insulin-like signaling. S597 also selectively activates IR signaling among different tissues and triggers IR endocytosis in the liver. Overall, our structural and functional studies guide future efforts to develop insulin mimetics targeting insulin resistance caused by defects in insulin binding and stabilization of insulin-activated state of IR, demonstrating the potential of structure-based drug design for insulin-resistant diseases.

Impact of the COVID-19 pandemic on congenital diaphragmatic hernia patients: a single-center retrospective study.

PURPOSE: To investigate the impact of COVID-19 on the treatment of children with congenital diaphragmatic hernia (CDH). METHODS: We retrospectively collected and compared the data of patients with CDH admitted between January 1, 2020 and December 31, 2021(study group) with the CDH patients admitted before the pandemic between January 1, 2018 and December 31, 2019 (control group). RESULTS: During the pandemic, 41 patients with CDH diagnosed prenatally were transferred to our hospital, and 40 underwent surgical repair. The number of patients treated in our hospital increased by 24.2% compared with the 33 patients before the pandemic. During the pandemic, the overall survival rate, postoperative survival rate and recurrence rate were 85.4%, 87.5% and 7.3%, respectively, and there were no significant differences compared with the control group (75.8%, 83.3% and 9.1%, respectively). The average length of hospital stay in patients admitted during the pandemic was longer than that in the control group (31 days vs. 16 days, P < 0.001), and the incidence of nosocomial infection was higher than that in the control group (19.5% vs. 3%, P = 0.037). CONCLUSIONS: CDH patients confirmed to be SARS-CoV-2 infection-free can receive routine treatment. Our data indicate that the implementation of protective measures during the COVID-19 pandemic, along with appropriate screening and case evaluation, do not have a negative impact on the prognosis of children.

Activation of STING by targeting a pocket in the transmembrane domain.

Stimulator of interferon genes (STING) is an adaptor protein in innate immunity against DNA viruses or bacteria1-5. STING-mediated immunity could be exploited in the development of vaccines or cancer immunotherapies. STING is a transmembrane dimeric protein that is located in the endoplasmic reticulum or in the Golgi apparatus. STING is activated by the binding of its cytoplasmic ligand-binding domain to cyclic dinucleotides that are produced by the DNA sensor cyclic GMP-AMP (cGAMP) synthase or by invading bacteria1,6,7. Cyclic dinucleotides induce a conformational change in the STING ligand-binding domain, which leads to a high-order oligomerization of STING that is essential for triggering the downstream signalling pathways8,9. However, the cGAMP-induced STING oligomers tend to dissociate in solution and have not been resolved to high resolution, which limits our understanding of the activation mechanism. Here we show that a small-molecule agonist, compound 53 (C53)10, promotes the oligomerization and activation of human STING through a mechanism orthogonal to that of cGAMP. We determined a cryo-electron microscopy structure of STING bound to both C53 and cGAMP, revealing a stable oligomer that is formed by side-by-side packing and has a curled overall shape. Notably, C53 binds to a cryptic pocket in the STING transmembrane domain, between the two subunits of the STING dimer. This binding triggers outward shifts of transmembrane helices in the dimer, and induces inter-dimer interactions between these helices to mediate the formation of the high-order oligomer. Our functional analyses show that cGAMP and C53 together induce stronger activation of STING than either ligand alone.

A structural model of a Ras-Raf signalosome.

The protein K-Ras functions as a molecular switch in signaling pathways regulating cell growth. In the human mitogen-activated protein kinase (MAPK) pathway, which is implicated in many cancers, multiple K-Ras proteins are thought to assemble at the cell membrane with Ras effector proteins from the Raf family. Here we propose an atomistic structural model for such an assembly. Our starting point was an asymmetric guanosine triphosphate-mediated K-Ras dimer model, which we generated using unbiased molecular dynamics simulations and verified with mutagenesis experiments. Adding further K-Ras monomers in a head-to-tail fashion led to a compact helical assembly, a model we validated using electron microscopy and cell-based experiments. This assembly stabilizes K-Ras in its active state and presents composite interfaces to facilitate Raf binding. Guided by existing experimental data, we then positioned C-Raf, the downstream kinase MEK1 and accessory proteins (Galectin-3 and 14-3-3σ) on and around the helical assembly. The resulting Ras-Raf signalosome model offers an explanation for a large body of data on MAPK signaling.

Structural basis of the activation of c-MET receptor.

The c-MET receptor is a receptor tyrosine kinase (RTK) that plays essential roles in normal cell development and motility. Aberrant activation of c-MET can lead to both tumors growth and metastatic progression of cancer cells. C-MET can be activated by either hepatocyte growth factor (HGF), or its natural isoform NK1. Here, we report the cryo-EM structures of c-MET/HGF and c-MET/NK1 complexes in the active state. The c-MET/HGF complex structure reveals that, by utilizing two distinct interfaces, one HGF molecule is sufficient to induce a specific dimerization mode of c-MET for receptor activation. The binding of heparin as well as a second HGF to the 2:1 c-MET:HGF complex further stabilize this active conformation. Distinct to HGF, NK1 forms a stable dimer, and bridges two c-METs in a symmetrical manner for activation. Collectively, our studies provide structural insights into the activation mechanisms of c-MET, and reveal how two isoforms of the same ligand use dramatically different mechanisms to activate the receptor.

Structural and Mechanistic Regulation of the Pro-degenerative NAD Hydrolase SARM1.

The NADase SARM1 is a central switch in injury-activated axon degeneration, an early hallmark of many neurological diseases. Here, we present cryo-electron microscopy (cryo-EM) structures of autoinhibited (3.3 Å) and active SARM1 (6.8 Å) and provide mechanistic insight into the tight regulation of SARM1's function by the local metabolic environment. Although both states retain an octameric core, the defining feature of the autoinhibited state is a lock between the autoinhibitory Armadillo/HEAT motif (ARM) and catalytic Toll/interleukin-1 receptor (TIR) domains, which traps SARM1 in an inactive state. Mutations that break this lock activate SARM1, resulting in catastrophic neuronal death. Notably, the mutants cannot be further activated by the endogenous activator nicotinamide mononucleotide (NMN), and active SARM1 is product inhibited by Nicotinamide (NAM), highlighting SARM1's functional dependence on key metabolites in the NAD salvage pathway. Our studies provide a molecular understanding of SARM1's transition from an autoinhibited to an injury-activated state and lay the foundation for future SARM1-based therapies to treat axonopathies.

Structures and Mechanisms in the cGAS-STING Innate Immunity Pathway.

Besides its role as the blueprint of life, DNA can also alert the cell to the presence of microbial pathogens as well as damaged or malignant cells. A major sensor of DNA that triggers the innate immune response is cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) (cGAMP) synthase (cGAS), which produces the second messenger cGAMP. cGAMP activates stimulator of interferon genes (STING), which activates a signaling cascade leading to the production of type I interferons and other immune mediators. Recent research has demonstrated an expanding role of the cGAS-cGAMP-STING pathway in many physiological and pathological processes, including host defense against microbial infections, anti-tumor immunity, cellular senescence, autophagy, and autoimmune and inflammatory diseases. Biochemical and structural studies have elucidated the mechanism of signal transduction in the cGAS pathway at the atomic resolution. This review focuses on the structural and mechanistic insights into the roles of cGAS and STING in immunity and diseases revealed by these recent studies.

Cryo-EM structure of the human cohesin-NIPBL-DNA complex.

As a ring-shaped adenosine triphosphatase (ATPase) machine, cohesin organizes the eukaryotic genome by extruding DNA loops and mediates sister chromatid cohesion by topologically entrapping DNA. How cohesin executes these fundamental DNA transactions is not understood. Using cryo-electron microscopy (cryo-EM), we determined the structure of human cohesin bound to its loader NIPBL and DNA at medium resolution. Cohesin and NIPBL interact extensively and together form a central tunnel to entrap a 72-base pair DNA. NIPBL and DNA promote the engagement of cohesin's ATPase head domains and ATP binding. The hinge domains of cohesin adopt an "open washer" conformation and dock onto the STAG1 subunit. Our structure explains the synergistic activation of cohesin by NIPBL and DNA and provides insight into DNA entrapment by cohesin.

Intranasal Delivery of Bone Marrow Stromal Cells Preconditioned with Fasudil to Treat a Mouse Model of Parkinson's Disease.

OBJECTIVE: Stem cell transplantation is a promising strategy with great potential to treat Parkinson's disease (PD). Nevertheless, improving the cell delivery route and optimising implanted cells are necessary to increase the therapeutic effect. Herein, we investigated whether intranasal delivery of bone marrow stromal cells (BMSCs) has beneficial effects in a PD mouse model and whether the therapeutic potential of BMSCs could be enhanced by preconditioning with fasudil. METHODS: A PD mouse model was developed by intraperitoneally administering 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Mice were treated intranasally with phosphate buffered saline (PBS), BMSCs, or BMSCs preconditioned with fasudil. One month later, the effects of BMSC treatment were analysed. RESULTS: Our study showed that fasudil could accelerate the proliferation of BMSCs and promote brain-derived neurotrophic factor (BDNF) secretion in vitro. Intranasally administered BMSCs were capable of surviving and migrating in the brain. Intranasal delivery of BMSCs preconditioned with fasudil significantly improved motor function and reduced dopaminergic neuron loss in substantia nigra; treatment with BMSCs and PBS resulted in similar outcomes. Preconditioning with fasudil inhibited the activation and aggregation of microglia, suppressed immune response, and reinforced BDNF secretion in MPTP-PD mice significantly more than treatment with BMSCs alone. CONCLUSION: The present study demonstrates that intranasally administering BMSCs preconditioned with fasudil is a promising cell-based therapy for PD.

Cryo-EM analyses reveal the common mechanism and diversification in the activation of RET by different ligands.

RET is a receptor tyrosine kinase (RTK) that plays essential roles in development and has been implicated in several human diseases. Different from most of RTKs, RET requires not only its cognate ligands but also co-receptors for activation, the mechanisms of which remain unclear due to lack of high-resolution structures of the ligand/co-receptor/receptor complexes. Here, we report cryo-EM structures of the extracellular region ternary complexes of GDF15/GFRAL/RET, GDNF/GFRα1/RET, NRTN/GFRα2/RET and ARTN/GFRα3/RET. These structures reveal that all the four ligand/co-receptor pairs, while using different atomic interactions, induce a specific dimerization mode of RET that is poised to bring the two kinase domains into close proximity for cross-phosphorylation. The NRTN/GFRα2/RET dimeric complex further pack into a tetrameric assembly, which is shown by our cell-based assays to regulate the endocytosis of RET. Our analyses therefore reveal both the common mechanism and diversification in the activation of RET by different ligands.

Structural basis of STING binding with and phosphorylation by TBK1.

The invasion of mammalian cytoplasm by microbial DNA from infectious pathogens or by self DNA from the nucleus or mitochondria represents a danger signal that alerts the host immune system1. Cyclic GMP-AMP synthase (cGAS) is a sensor of cytoplasmic DNA that activates the type-I interferon pathway2. On binding to DNA, cGAS is activated to catalyse the synthesis of cyclic GMP-AMP (cGAMP) from GTP and ATP3. cGAMP functions as a second messenger that binds to and activates stimulator of interferon genes (STING)3-9. STING then recruits and activates tank-binding kinase 1 (TBK1), which phosphorylates STING and the transcription factor IRF3 to induce type-I interferons and other cytokines10,11. However, how cGAMP-bound STING activates TBK1 and IRF3 is not understood. Here we present the cryo-electron microscopy structure of human TBK1 in complex with cGAMP-bound, full-length chicken STING. The structure reveals that the C-terminal tail of STING adopts a ß-strand-like conformation and inserts into a groove between the kinase domain of one TBK1 subunit and the scaffold and dimerization domain of the second subunit in the TBK1 dimer. In this binding mode, the phosphorylation site Ser366 in the STING tail cannot reach the kinase-domain active site of bound TBK1, which suggests that STING phosphorylation by TBK1 requires the oligomerization of both proteins. Mutational analyses validate the interaction mode between TBK1 and STING and support a model in which high-order oligomerization of STING and TBK1, induced by cGAMP, leads to STING phosphorylation by TBK1.

Cryo-EM structures of STING reveal its mechanism of activation by cyclic GMP-AMP.

Infections by pathogens that contain DNA trigger the production of type-I interferons and inflammatory cytokines through cyclic GMP-AMP synthase, which produces 2'3'-cyclic GMP-AMP (cGAMP) that binds to and activates stimulator of interferon genes (STING; also known as TMEM173, MITA, ERIS and MPYS)1-8. STING is an endoplasmic-reticulum membrane protein that contains four transmembrane helices followed by a cytoplasmic ligand-binding and signalling domain9-13. The cytoplasmic domain of STING forms a dimer, which undergoes a conformational change upon binding to cGAMP9,14. However, it remains unclear how this conformational change leads to STING activation. Here we present cryo-electron microscopy structures of full-length STING from human and chicken in the inactive dimeric state (about 80 kDa in size), as well as cGAMP-bound chicken STING in both the dimeric and tetrameric states. The structures show that the transmembrane and cytoplasmic regions interact to form an integrated, domain-swapped dimeric assembly. Closure of the ligand-binding domain, induced by cGAMP, leads to a 180° rotation of the ligand-binding domain relative to the transmembrane domain. This rotation is coupled to a conformational change in a loop on the side of the ligand-binding-domain dimer, which leads to the formation of the STING tetramer and higher-order oligomers through side-by-side packing. This model of STING oligomerization and activation is supported by our structure-based mutational analyses.

Cryo-EM structure of the adenosine A2A receptor coupled to an engineered heterotrimeric G protein.

The adenosine A2A receptor (A2AR) is a prototypical G protein-coupled receptor (GPCR) that couples to the heterotrimeric G protein GS. Here, we determine the structure by electron cryo-microscopy (cryo-EM) of A2AR at pH 7.5 bound to the small molecule agonist NECA and coupled to an engineered heterotrimeric G protein, which contains mini-GS, the ßγ subunits and nanobody Nb35. Most regions of the complex have a resolution of ~3.8 Å or better. Comparison with the 3.4 Å resolution crystal structure shows that the receptor and mini-GS are virtually identical and that the density of the side chains and ligand are of comparable quality. However, the cryo-EM density map also indicates regions that are flexible in comparison to the crystal structures, which unexpectedly includes regions in the ligand binding pocket. In addition, an interaction between intracellular loop 1 of the receptor and the ß subunit of the G protein was observed.

Structures of the calcium-activated, non-selective cation channel TRPM4.

TRPM4 is a calcium-activated, phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) -modulated, non-selective cation channel that belongs to the family of melastatin-related transient receptor potential (TRPM) channels. Here we present the electron cryo-microscopy structures of the mouse TRPM4 channel with and without ATP. TRPM4 consists of multiple transmembrane and cytosolic domains, which assemble into a three-tiered architecture. The N-terminal nucleotide-binding domain and the C-terminal coiled-coil participate in the tetrameric assembly of the channel; ATP binds at the nucleotide-binding domain and inhibits channel activity. TRPM4 has an exceptionally wide filter but is only permeable to monovalent cations; filter residue Gln973 is essential in defining monovalent selectivity. The S1-S4 domain and the post-S6 TRP domain form the central gating apparatus that probably houses the Ca2+- and PtdIns(4,5)P2-binding sites. These structures provide an essential starting point for elucidating the complex gating mechanisms of TRPM4 and reveal the molecular architecture of the TRPM family.