Structure and topography of the synaptic V-ATPase-synaptophysin complex.

Synaptic vesicles are organelles with a precisely defined protein and lipid composition1,2, yet the molecular mechanisms for the biogenesis of synaptic vesicles are mainly unknown. Here, we discovered a well-defined interface between the synaptic vesicle V-ATPase and synaptophysin by in situ cryo-electron tomography and single particle cryo-electron microscopy of functional synaptic vesicles isolated from mouse brains3. The synaptic vesicle V-ATPase is an ATP-dependent proton pump that establishes the protein gradient across the synaptic vesicle, which in turn drives the uptake of neurotransmitters4,5. Synaptophysin6 and its paralogs synaptoporin7 and synaptogyrin8 belong to a family of abundant synaptic vesicle proteins whose function is still unclear. We performed structural and functional studies of synaptophysin knockout mice, confirming the identity of synaptophysin as an interaction partner with the V-ATPase. Although there is little change in the conformation of the V-ATPase upon interaction with synaptophysin, the presence of synaptophysin in synaptic vesicles profoundly affects the copy number of V-ATPases. This effect on the topography of synaptic vesicles suggests that synaptophysin assists in their biogenesis. In support of this model, we observed that synaptophysin knockout mice exhibit severe seizure susceptibility, suggesting an imbalance of neurotransmitter release as a physiological consequence of the absence of synaptophysin.

Reprogramming macrophage by targeting VEGF and CD40 potentiates OX40 immunotherapy.

The low clinical response rate of checkpoint blockades, such as PD-1 and CTLA-4, highlighted the requirements of agonistic antibodies to boost optimal T cell responses. OX40, a co-stimulatory receptor on the T cells, plays a crucial role in promoting T cell survival and differentiation. However, the clinical efficacy of anti-OX40 agonistic antibodies was unimpressive. To explore the mechanism underlying the action of anti-OX40 agonists to improve the anti-tumor efficacy, we analyzed the dynamic changes of tumor-infiltrating immune cells at different days post-treatments using single-cell RNA-sequencing (scRNA-seq). In this study, we found that tumor-infiltrating regulatory T (Treg) cells were reduced after two rounds of anti-OX40 treatment, but the increase of infiltration and activation of CD8+ effector T cells, as well as M1 polarization in the tumor were only observed after three rounds of treatments. Moreover, our group first analyzed the antitumor effect of anti-OX40 treatments on regulating the macrophages and discovered the dynamic changes of vascular endothelial growth factor (VEGF) and CD40 signaling pathways on macrophages, indicating their possibility to being potential combination targets to improve the anti-OX40 agonists efficacy. The combination of VEGFR inhibitors or anti-CD40 agonist antibody with anti-OX40 agonists exhibited more remarkable inhibition of tumor growth. Therefore, the mechanism-driven combination of anti-OX40 agonists with VEGFR inhibitors or anti-CD40 agonists represented promising strategies.

Structure-based design of a SARS-CoV-2 Omicron-specific inhibitor.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) introduced a relatively large number of mutations, including three mutations in the highly conserved heptad repeat 1 (HR1) region of the spike glycoprotein (S) critical for its membrane fusion activity. We show that one of these mutations, N969K induces a substantial displacement in the structure of the heptad repeat 2 (HR2) backbone in the HR1HR2 postfusion bundle. Due to this mutation, fusion-entry peptide inhibitors based on the Wuhan strain sequence are less efficacious. Here, we report an Omicron-specific peptide inhibitor designed based on the structure of the Omicron HR1HR2 postfusion bundle. Specifically, we inserted an additional residue in HR2 near the Omicron HR1 K969 residue to better accommodate the N969K mutation and relieve the distortion in the structure of the HR1HR2 postfusion bundle it introduced. The designed inhibitor recovers the loss of inhibition activity of the original longHR2_42 peptide with the Wuhan strain sequence against the Omicron variant in both a cell-cell fusion assay and a vesicular stomatitis virus (VSV)-SARS-CoV-2 chimera infection assay, suggesting that a similar approach could be used to combat future variants. From a mechanistic perspective, our work suggests the interactions in the extended region of HR2 may mediate the initial landing of HR2 onto HR1 during the transition of the S protein from the prehairpin intermediate to the postfusion state.

Nanomolar inhibition of SARS-CoV-2 infection by an unmodified peptide targeting the prehairpin intermediate of the spike protein.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available coronavirus disease 2019 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors, which block formation of the so-called heptad repeat 1 and 2 (HR1HR2) six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. We performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based and virus-based assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ∼100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a prehairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the prehairpin intermediate of the S protein.

Nanomolar inhibition of SARS-CoV-2 infection by an unmodified peptide targeting the pre-hairpin intermediate of the spike protein.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available COVID-19 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors which block formation of the so-called HR1HR2 six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. Here we performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based fusion, VSV-SARS-CoV-2 chimera, and authentic SARS-CoV-2 infection assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ~100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a pre-hairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the pre-hairpin intermediate of the S protein. Significance Statement: SARS-CoV-2 infection requires fusion of viral and host membranes, mediated by the viral spike glycoprotein (S). Due to the importance of viral membrane fusion, S has been a popular target for developing vaccines and therapeutics. We discovered a simple peptide that inhibits infection by all major variants of SARS-CoV-2 with nanomolar efficacies. In marked contrast, widely used shorter peptides that lack a key N-terminal extension are about 100 x less potent than this peptide. Our results suggest that a simple peptide with a suitable sequence can be a potent and cost-effective therapeutic against COVID-19 and they provide new insights at the virus entry mechanism.

Structural conservation among variants of the SARS-CoV-2 spike postfusion bundle.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available COVID-19 vaccines and monoclonal antibody therapies due to structural and dynamic changes of the viral spike glycoprotein (S). The heptad repeat 1 (HR1) and heptad repeat 2 (HR2) domains of S drive virus­host membrane fusion by assembly into a six-helix bundle, resulting in delivery of viral RNA into the host cell. We surveyed mutations of currently reported SARS-CoV-2 variants and selected eight mutations, including Q954H, N969K, and L981F from the Omicron variant, in the postfusion HR1HR2 bundle for functional and structural studies. We designed a molecular scaffold to determine cryogenic electron microscopy (cryo-EM) structures of HR1HR2 at 2.2­3.8 Å resolution by linking the trimeric N termini of four HR1 fragments to four trimeric C termini of the Dps4 dodecamer from Nostoc punctiforme. This molecular scaffold enables efficient sample preparation and structure determination of the HR1HR2 bundle and its mutants by single-particle cryo-EM. Our structure of the wild-type HR1HR2 bundle resolves uncertainties in previously determined structures. The mutant structures reveal side-chain positions of the mutations and their primarily local effects on the interactions between HR1 and HR2. These mutations do not alter the global architecture of the postfusion HR1HR2 bundle, suggesting that the interfaces between HR1 and HR2 are good targets for developing antiviral inhibitors that should be efficacious against all known variants of SARS-CoV-2 to date. We also note that this work paves the way for similar studies in more distantly related viruses.

Inhibition of calcium-triggered secretion by hydrocarbon-stapled peptides.

Membrane fusion triggered by Ca2+ is orchestrated by a conserved set of proteins to mediate synaptic neurotransmitter release, mucin secretion and other regulated exocytic processes1-4. For neurotransmitter release, the Ca2+ sensitivity is introduced by interactions between the Ca2+ sensor synaptotagmin and the SNARE complex5, and sequence conservation and functional studies suggest that this mechanism is also conserved for mucin secretion6. Disruption of Ca2+-triggered membrane fusion by a pharmacological agent would have therapeutic value for mucus hypersecretion as it is the major cause of airway obstruction in the pathophysiology of respiratory viral infection, asthma, chronic obstructive pulmonary disease and cystic fibrosis7-11. Here we designed a hydrocarbon-stapled peptide that specifically disrupts Ca2+-triggered membrane fusion by interfering with the so-called primary interface between the neuronal SNARE complex and the Ca2+-binding C2B domain of synaptotagmin-1. In reconstituted systems with these neuronal synaptic proteins or with their airway homologues syntaxin-3, SNAP-23, VAMP8, synaptotagmin-2, along with Munc13-2 and Munc18-2, the stapled peptide strongly suppressed Ca2+-triggered fusion at physiological Ca2+ concentrations. Conjugation of cell-penetrating peptides to the stapled peptide resulted in efficient delivery into cultured human airway epithelial cells and mouse airway epithelium, where it markedly and specifically reduced stimulated mucin secretion in both systems, and substantially attenuated mucus occlusion of mouse airways. Taken together, peptides that disrupt Ca2+-triggered membrane fusion may enable the therapeutic modulation of mucin secretory pathways.

Structure of the translating Neurospora ribosome arrested by cycloheximide.

Ribosomes translate RNA into proteins. The protein synthesis inhibitor cycloheximide (CHX) is widely used to inhibit eukaryotic ribosomes engaged in translation elongation. However, the lack of structural data for actively translating polyribosomes stalled by CHX leaves unanswered the question of which elongation step is inhibited. We elucidated CHX's mechanism of action based on the cryo-electron microscopy structure of actively translating Neurospora crassa ribosomes bound with CHX at 2.7-Å resolution. The ribosome structure from this filamentous fungus contains clearly resolved ribosomal protein eL28, like higher eukaryotes but unlike budding yeast, which lacks eL28. Despite some differences in overall structures, the ribosomes from Neurospora, yeast, and humans all contain a highly conserved CHX binding site. We also sequenced classic Neurospora CHX-resistant alleles. These mutations, including one at a residue not previously observed to affect CHX resistance in eukaryotes, were in the large subunit proteins uL15 and eL42 that are part of the CHX-binding pocket. In addition to A-site transfer RNA (tRNA), P-site tRNA, messenger RNA, and CHX that are associated with the translating N. crassa ribosome, spermidine is present near the CHX binding site close to the E site on the large subunit. The tRNAs in the peptidyl transferase center are in the A/A site and the P/P site. The nascent peptide is attached to the A-site tRNA and not to the P-site tRNA. The structural and functional data obtained show that CHX arrests the ribosome in the classical PRE translocation state and does not interfere with A-site reactivity.

Hierarchical natural move Monte Carlo refines flexible RNA structures into cryo-EM densities.

Ribonucleic acids (RNAs) play essential roles in living cells. Many of them fold into defined three-dimensional (3D) structures to perform functions. Recent advances in single-particle cryo-electron microscopy (cryo-EM) have enabled structure determinations of RNA to atomic resolutions. However, most RNA molecules are structurally flexible, limiting the resolution of their structures solved by cryo-EM. In modeling these molecules, several computational methods are limited by the requirement of massive computational resources and/or the low efficiency in exploring large-scale structural variations. Here we use hierarchical natural move Monte Carlo (HNMMC), which takes advantage of collective motions for groups of nucleic acid residues, to refine RNA structures into their cryo-EM maps, preserving atomic details in the models. After validating the method on a simulated density map of tRNA, we applied it to objectively obtain the model of the folding intermediate for the specificity domain of ribonuclease P from Bacillus subtilis and refine a flexible ribosomal RNA (rRNA) expansion segment from the Mycobacterium tuberculosis (Mtb) ribosome in different conformational states. Finally, we used HNMMC to model atomic details and flexibility for two distinct conformations of the complete genomic RNA (gRNA) inside MS2, a single-stranded RNA virus, revealing multiple pathways for its capsid assembly.

Structural basis for the adsorption of a single-stranded RNA bacteriophage.

Single-stranded RNA bacteriophages (ssRNA phages) infect Gram-negative bacteria via a single maturation protein (Mat), which attaches to a retractile pilus of the host. Here we present structures of the ssRNA phage MS2 in complex with the Escherichia coli F-pilus, showing a network of hydrophobic and electrostatic interactions at the Mat-pilus interface. Moreover, binding of the pilus induces slight orientational variations of the Mat relative to the rest of the phage capsid, priming the Mat-connected genomic RNA (gRNA) for its release from the virions. The exposed tip of the attached Mat points opposite to the direction of the pilus retraction, which may facilitate the translocation of the gRNA from the capsid into the host cytosol. In addition, our structures determine the orientation of the assembled F-pilin subunits relative to the cell envelope, providing insights into the F-like type IV secretion systems.

Structural insights into species-specific features of the ribosome from the human pathogen Mycobacterium tuberculosis.

Ribosomes from Mycobacterium tuberculosis (Mtb) possess species-specific ribosomal RNA (rRNA) expansion segments and ribosomal proteins (rProtein). Here, we present the near-atomic structures of the Mtb 50S ribosomal subunit and the complete Mtb 70S ribosome, solved by cryo-electron microscopy. Upon joining of the large and small ribosomal subunits, a 100-nt long expansion segment of the Mtb 23S rRNA, named H54a or the 'handle', switches interactions from with rRNA helix H68 and rProtein uL2 to with rProtein bS6, forming a new intersubunit bridge 'B9'. In Mtb 70S, bridge B9 is mostly maintained, leading to correlated motions among the handle, the L1 stalk and the small subunit in the rotated and non-rotated states. Two new protein densities were discovered near the decoding center and the peptidyl transferase center, respectively. These results provide a structural basis for studying translation in Mtb as well as developing new tuberculosis drugs.

Dynamic Interactions of Arabidopsis TEN1: Stabilizing Telomeres in Response to Heat Stress.

Telomeres are the essential nucleoprotein structures that provide a physical cap for the ends of linear chromosomes. The highly conserved CST (CTC1/STN1/TEN1) protein complex facilitates telomeric DNA replication and promotes telomere stability. Here we report three unexpected properties of Arabidopsis thaliana TEN1 that indicate it possesses functions distinct from other previously characterized telomere proteins. First, we show that telomeres in ten1 mutants are highly sensitive to thermal stress. Heat shock causes abrupt and dramatic loss of telomeric DNA in ten1 plants, likely via deletional recombination. Second, we show that AtTEN1 has the properties of a heat-shock induced molecular chaperone. At elevated temperature, AtTEN1 rapidly assembles into high molecular weight homo-oligomeric complexes that efficiently suppress heat-induced aggregation of model protein substrates in vitro. Finally, we report that AtTEN1 specifically protects CTC1 from heat-induced aggregation in vitro, and from heat-induced protein degradation and loss of telomere association in vivo. Collectively, these observations define Arabidopsis TEN1 as a highly dynamic protein that works in concert with CTC1 to preserve telomere integrity in response to environmental stress.

Structures of the Carbon-Phosphorus Lyase Complex Reveal the Binding Mode of the NBD-like PhnK.

The carbon-phosphorus (C-P) lyase complex is essential for the metabolism of unactivated phosphonates to phosphate in bacteria. Using single-particle cryo-electron microscopy, we determined two structures of the C-P lyase core complex PhnG2H2I2J2, with or without PhnK. PhnG2H2I2J2 is a two-fold symmetric hetero-octamer. Its two PhnJ subunits provide two identical binding sites for PhnK. Only one PhnK binds to PhnG2H2I2J2 due to steric hindrance. PhnK is homologous to the nucleotide-binding domain (NBD) of ATP-binding cassette transporters. The α helices 3 and 4 of PhnK bind to α helix 6 and a loop (residues 227-230) of PhnJ, in a different mode from the binding of NBDs to their transmembrane partners. Moreover, binding of PhnK exposes the active site residue, Gly32 of PhnJ, located near the interface between PhnJ and PhnH. This structural information provides a basis for further deciphering of the reaction mechanism of the C-P lyase.

Structure of Ribosomal Silencing Factor Bound to Mycobacterium tuberculosis Ribosome.

The ribosomal silencing factor RsfS slows cell growth by inhibiting protein synthesis during periods of diminished nutrient availability. The crystal structure of Mycobacterium tuberculosis (Mtb) RsfS, together with the cryo-electron microscopy (EM) structure of the large subunit 50S of Mtb ribosome, reveals how inhibition of protein synthesis by RsfS occurs. RsfS binds to the 50S at L14, which, when occupied, blocks the association of the small subunit 30S. Although Mtb RsfS is a dimer in solution, only a single subunit binds to 50S. The overlap between the dimer interface and the L14 binding interface confirms that the RsfS dimer must first dissociate to a monomer in order to bind to L14. RsfS interacts primarily through electrostatic and hydrogen bonding to L14. The EM structure shows extended rRNA density that it is not found in the Escherichia coli ribosome, the most striking of these being the extended RNA helix of H54a.

Multiple metabolic requirements for size homeostasis and initiation of division in Saccharomyces cerevisiae.

Most cells must grow before they can divide, but it is not known how cells determine when they have grown enough so they can commit to a new round of cell division. Several parameters affect the timing of initiation of division: cell size at birth, the size cells have to reach when they commit to division, and how fast they reach that size. We report that Saccharomyces cerevisiae mutants in metabolic and biosynthetic pathways differ in these variables, controlling the timing of initiation of cell division in various ways. Some mutants affect the size at birth, size at initiation of division, the rate of increase in size, or any combination of the above. Furthermore, we show that adenylate kinase, encoded by ADK1, is a significant determinant of the efficiency of size control mechanisms. Finally, our data argue strongly that the cell size at division is not necessarily a function of the rate cells increase in size in the G1 phase of the cell cycle. Taken together, these findings reveal an unexpected diversity in the G1 cell cycle phenotypes of metabolic and biosynthetic mutants, suggesting that growth requirements for cell division are multiple, distinct and imposed throughout the G1 phase of the cell cycle.