Activation of the insulin receptor by insulin-like growth factor 2.

Insulin receptor (IR) controls growth and metabolism. Insulin-like growth factor 2 (IGF2) has different binding properties on two IR isoforms, mimicking insulin's function. However, the molecular mechanism underlying IGF2-induced IR activation remains unclear. Here, we present cryo-EM structures of full-length human long isoform IR (IR-B) in both the inactive and IGF2-bound active states, and short isoform IR (IR-A) in the IGF2-bound active state. Under saturated IGF2 concentrations, both the IR-A and IR-B adopt predominantly asymmetric conformations with two or three IGF2s bound at site-1 and site-2, which differs from that insulin saturated IR forms an exclusively T-shaped symmetric conformation. IGF2 exhibits a relatively weak binding to IR site-2 compared to insulin, making it less potent in promoting full IR activation. Cell-based experiments validated the functional importance of IGF2 binding to two distinct binding sites in optimal IR signaling and trafficking. In the inactive state, the C-terminus of α-CT of IR-B contacts FnIII-2 domain of the same protomer, hindering its threading into the C-loop of IGF2, thus reducing the association rate of IGF2 with IR-B. Collectively, our studies demonstrate the activation mechanism of IR by IGF2 and reveal the molecular basis underlying the different affinity of IGF2 to IR-A and IR-B.

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.

Structural basis of insulin fibrillation.

Insulin is a hormone responsible for maintaining normal glucose levels by activating insulin receptor (IR) and is the primary treatment for diabetes. However, insulin is prone to unfolding and forming cross-ß fibers. Fibrillation complicates insulin storage and therapeutic application. Molecular details of insulin fibrillation remain unclear, hindering efforts to prevent fibrillation process. Here, we characterized insulin fibrils using cryo-electron microscopy (cryo-EM), showing multiple forms that contain one or more of the protofilaments containing both the A and B chains of insulin linked by disulfide bonds. We solved the cryo-EM structure of one of the fibril forms composed of two protofilaments at 3.2-Å resolution, which reveals both the ß sheet conformation of the protofilament and the packing interaction between them that underlie the fibrillation. On the basis of this structure, we designed several insulin mutants that display reduced fibrillation while maintaining native IR signaling activity. These designed insulin analogs may be developed into more effective therapeutics for type 1 diabetes.

CTCF and R-loops are boundaries of cohesin-mediated DNA looping.

Cohesin and CCCTC-binding factor (CTCF) are key regulatory proteins of three-dimensional (3D) genome organization. Cohesin extrudes DNA loops that are anchored by CTCF in a polar orientation. Here, we present direct evidence that CTCF binding polarity controls cohesin-mediated DNA looping. Using single-molecule imaging, we demonstrate that a critical N-terminal motif of CTCF blocks cohesin translocation and DNA looping. The cryo-EM structure of the cohesin-CTCF complex reveals that this CTCF motif ahead of zinc fingers can only reach its binding site on the STAG1 cohesin subunit when the N terminus of CTCF faces cohesin. Remarkably, a C-terminally oriented CTCF accelerates DNA compaction by cohesin. DNA-bound Cas9 and Cas12a ribonucleoproteins are also polar cohesin barriers, indicating that stalling may be intrinsic to cohesin itself. Finally, we show that RNA-DNA hybrids (R-loops) block cohesin-mediated DNA compaction in vitro and are enriched with cohesin subunits in vivo, likely forming TAD boundaries.

Structural insights into the assembly of the agrin/LRP4/MuSK signaling complex.

MuSK is a receptor tyrosine kinase (RTK) that plays essential roles in the formation and maintenance of the neuromuscular junction. Distinct from most members of RTK family, MuSK activation requires not only its cognate ligand agrin but also its coreceptors LRP4. However, how agrin and LRP4 coactivate MuSK remains unclear. Here, we report the cryo-EM structure of the extracellular ternary complex of agrin/LRP4/MuSK in a stoichiometry of 1:1:1. This structure reveals that arc-shaped LRP4 simultaneously recruits both agrin and MuSK to its central cavity, thereby promoting a direct interaction between agrin and MuSK. Our cryo-EM analyses therefore uncover the assembly mechanism of agrin/LRP4/MuSK signaling complex and reveal how MuSK receptor is activated by concurrent binding of agrin and LRP4.

Inhibition of FAM46/TENT5 activity by BCCIPα adopting a unique fold.

The FAM46 (also known as TENT5) proteins are noncanonical poly(A) polymerases (PAPs) implicated in regulating RNA stability. The regulatory mechanisms of FAM46 are poorly understood. Here, we report that the nuclear protein BCCIPα, but not the alternatively spliced isoform BCCIPß, binds FAM46 and inhibits their PAP activity. Unexpectedly, our structures of the FAM46A/BCCIPα and FAM46C/BCCIPα complexes show that, despite sharing most of the sequence and differing only at the C-terminal portion, BCCIPα adopts a unique structure completely different from BCCIPß. The distinct C-terminal segment of BCCIPα supports the adoption of the unique fold but does not directly interact with FAM46. The ß sheets in BCCIPα and FAM46 pack side by side to form an extended ß sheet. A helix-loop-helix segment in BCCIPα inserts into the active site cleft of FAM46, thereby inhibiting the PAP activity. Our results together show that the unique fold of BCCIPα underlies its interaction with and functional regulation of FAM46.

Structural basis of the alkaline pH-dependent activation of insulin receptor-related receptor.

The insulin receptor (IR) family is a subfamily of receptor tyrosine kinases that controls metabolic homeostasis and cell growth. Distinct from IR and insulin-like growth factor 1 receptor, whose activation requires ligand binding, insulin receptor-related receptor (IRR)-the third member of the IR family-is activated by alkaline pH. However, the molecular mechanism underlying alkaline pH-induced IRR activation remains unclear. Here, we present cryo-EM structures of human IRR in both neutral pH inactive and alkaline pH active states. Combined with mutagenesis and cellular assays, we show that, upon pH increase, electrostatic repulsion of the pH-sensitive motifs of IRR disrupts its autoinhibited state and promotes a scissor-like rotation between two protomers, leading to a T-shaped active conformation. Together, our study reveals an unprecedented alkaline pH-dependent activation mechanism of IRR, opening up opportunities to understand the structure-function relationship of this important receptor.

Association between Respiratory Function and Motor Function in Different Stages of Parkinson's Disease.

INTRODUCTION: Respiratory dysfunction in patients with Parkinson's disease (PD) could present in the early stage and worsen in the late stages. These changes could be a factor affecting the ability of daily living and quality of life of patients with PD. The primary objective of this study was to assess the respiratory function and its association with motor function in patients with different stages of PD. METHODS: This was a cross-sectional study conducted at the Huashan Hospital of Fudan University in Shanghai, China. The study included 65 patients diagnosed with PD (the Hoehn and Yahr scale between 1 and 4) and 20 healthy individuals of similar age, gender, weight, and height. The ventilatory function was assessed using the spirometry. Motor function was evaluated using subscale III of the United Parkinson's disease rating scale (UPDRS-III). After confirming the normality of data distribution, we performed one-way ANOVA with a Tukey's post hoc test. RESULTS: Compared with the healthy individuals, there was no statistical significance in forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), and forced expiratory volume in 1 s/forced vital capacity (FEV1/FVC) in the H&Y 1 group and H&Y 2 group (p > 0.05) but reduced peak expiratory flow (PEF) in the H&Y 2 group (p = 0.002). Reduced FVC, FEV1, and PEF was seen in the H&Y 3 group (p = 0.002, p = 0.001, and p = 0.0001, respectively). Reduced FVC, FEV1, PEF, and FEF25-75% was seen in the H&Y 4 group (p = 0.001, p = 0.0001, p = 0.0001, and p = 0.025, respectively). The correlation analysis revealed that there was a significant negative correlation between FVC and UPDRS-III scores (r = -0.248, p = 0.046), disease duration (r = -0.276, p = 0.026), H&Y scale (r = -0.415, p = 0.001). FEV1 was negatively correlated with UPDRS-III scores (r = -0.277, p = 0.025), disease duration (r = -0.291, p = 0.019), H&Y scale (r = -0.434, p = 0.0001). FEF25-75% was negatively correlated with disease duration (r = -0.247, p = 0.047), H&Y scale (r = -0.278, p = 0.025). CONCLUSION: Our findings revealed that respiratory impairment is present in moderate and advanced PD patients, and directly related to the severity of the disease. It is important to conduct respiratory function test in the clinical practice.

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.

Molecular basis for the role of disulfide-linked αCTs in the activation of insulin-like growth factor 1 receptor and insulin receptor.

The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) control metabolic homeostasis and cell growth and proliferation. The IR and IGF1R form similar disulfide bonds linked homodimers in the apo-state; however, their ligand binding properties and the structures in the active state differ substantially. It has been proposed that the disulfide-linked C-terminal segment of α-chain (αCTs) of the IR and IGF1R control the cooperativity of ligand binding and regulate the receptor activation. Nevertheless, the molecular basis for the roles of disulfide-linked αCTs in IR and IGF1R activation are still unclear. Here, we report the cryo-EM structures of full-length mouse IGF1R/IGF1 and IR/insulin complexes with modified αCTs that have increased flexibility. Unlike the Γ-shaped asymmetric IGF1R dimer with a single IGF1 bound, the IGF1R with the enhanced flexibility of αCTs can form a T-shaped symmetric dimer with two IGF1s bound. Meanwhile, the IR with non-covalently linked αCTs predominantly adopts an asymmetric conformation with four insulins bound, which is distinct from the T-shaped symmetric IR. Using cell-based experiments, we further showed that both IGF1R and IR with the modified αCTs cannot activate the downstream signaling potently. Collectively, our studies demonstrate that the certain structural rigidity of disulfide-linked αCTs is critical for optimal IR and IGF1R signaling activation.

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.

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.

An antibody that neutralizes SARS-CoV-1 and SARS-CoV-2 by binding to a conserved spike epitope outside the receptor binding motif.

The rapid evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), such as the Omicron variants that are highly transmissible and immune evasive, underscores the need to develop therapeutic antibodies with broad neutralizing activities. Here, we used the LIBRA-seq technology, which identified SARS-CoV-2-specific B cells via DNA barcoding and subsequently single-cell sequenced BCRs, to identify an antibody, SW186, which could neutralize major SARS-CoV-2 variants of concern, including Beta, Delta, and Omicron, as well as SARS-CoV-1. The cryo-EM structure of SW186 bound to the receptor binding domain (RBD) of the viral spike protein showed that SW186 interacted with an epitope of the RBD that is not at the interface of its binding to the ACE2 receptor but is highly conserved among SARS coronaviruses. This epitope encompasses a glycosylation site (N343) of the viral spike protein. Administration of SW186 in mice after they were infected with SARS-CoV-2 Alpha, Beta, or Delta variants reduced the viral loads in the lung. These results demonstrated that SW186 neutralizes diverse SARS coronaviruses by binding to a conserved RBD epitope, which could serve as a target for further antibody development.

Cryo-electron Microscopic Analysis of Single-Pass Transmembrane Receptors.

Single-pass transmembrane receptors (SPTMRs) represent a diverse group of integral membrane proteins that are involved in many essential cellular processes, including signal transduction, cell adhesion, and transmembrane transport of materials. Dysregulation of the SPTMRs is linked with many human diseases. Despite extensive efforts in past decades, the mechanisms of action of the SPTMRs remain incompletely understood. One major hurdle is the lack of structures of the full-length SPTMRs in different functional states. Such structural information is difficult to obtain by traditional structural biology methods such as X-ray crystallography and nuclear magnetic resonance (NMR). The recent rapid development of single-particle cryo-electron microscopy (cryo-EM) has led to an exponential surge in the number of high-resolution structures of integral membrane proteins, including SPTMRs. Cryo-EM structures of SPTMRs solved in the past few years have tremendously improved our understanding of how SPTMRs function. In this review, we will highlight these progresses in the structural studies of SPTMRs by single-particle cryo-EM, analyze important structural details of each protein involved, and discuss their implications on the underlying mechanisms. Finally, we also briefly discuss remaining challenges and exciting opportunities in the field.

SNARE assembly enlightened by cryo-EM structures of a synaptobrevin-Munc18-1-syntaxin-1 complex.

Munc18-1 forms a template to organize assembly of the neuronal SNARE complex that triggers neurotransmitter release, binding first to a closed conformation of syntaxin-1 where its amino-terminal region interacts with the SNARE motif, and later binding to synaptobrevin. However, the mechanism of SNARE complex assembly remains unclear. Here, we report two cryo-EM structures of Munc18-1 bound to cross-linked syntaxin-1 and synaptobrevin. The structures allow visualization of how syntaxin-1 opens and reveal how part of the syntaxin-1 amino-terminal region can help nucleate interactions between the amino termini of the syntaxin-1 and synaptobrevin SNARE motifs, while their carboxyl termini bind to distal sites of Munc18-1. These observations, together with mutagenesis, SNARE complex assembly experiments, and fusion assays with reconstituted proteoliposomes, support a model whereby these interactions are critical to initiate SNARE complex assembly and multiple energy barriers enable diverse mechanisms for exquisite regulation of neurotransmitter release.

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.

Synergistic activation of the insulin receptor via two distinct sites.

Insulin receptor (IR) signaling controls multiple facets of animal physiology. Maximally four insulins bind to IR at two distinct sites, termed site-1 and site-2. However, the precise functional roles of each binding event during IR activation remain unresolved. Here, we showed that IR incompletely saturated with insulin predominantly forms an asymmetric conformation and exhibits partial activation. IR with one insulin bound adopts a Γ-shaped conformation. IR with two insulins bound assumes a Ƭ-shaped conformation. One insulin binds at site-1 and another simultaneously contacts both site-1 and site-2 in the Ƭ-shaped IR dimer. We further show that concurrent binding of four insulins to sites-1 and -2 prevents the formation of asymmetric IR and promotes the T-shaped symmetric, fully active state. Collectively, our results demonstrate how the synergistic binding of multiple insulins promotes optimal IR activation.

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.