Wah Chiu, Postdoctoral Faculty Sponsor
Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against type I-F CRISPR-Cas complex revealed by cryo-EM.
Proceedings of the National Academy of Sciences of the United States of America
Prokaryotes and viruses have fought a long battle against each other. Prokaryotes use CRISPR-Cas-mediated adaptive immunity, while conversely, viruses evolve multiple anti-CRISPR (Acr) proteins to defeat these CRISPR-Cas systems. The type I-F CRISPR-Cas system in Pseudomonas aeruginosa requires the crRNA-guided surveillance complex (Csy complex) to recognize the invading DNA. Although some Acr proteins against the Csy complex have been reported, other relevant Acr proteins still need studies to understand their mechanisms. Here, we obtain three structures of previously unresolved Acr proteins (AcrF9, AcrF8, and AcrF6) bound to the Csy complex using electron cryo-microscopy (cryo-EM), with resolution at 2.57 A, 3.42 A, and 3.15 A, respectively. The 2.57-A structure reveals fine details for each molecular component within the Csy complex as well as the direct and water-mediated interactions between proteins and CRISPR RNA (crRNA). Our structures also show unambiguously how these Acr proteins bind differently to the Csy complex. AcrF9 binds to key DNA-binding sites on the Csy spiral backbone. AcrF6 binds at the junction between Cas7.6f and Cas8f, which is critical for DNA duplex splitting. AcrF8 binds to a distinct position on the Csy spiral backbone and forms interactions with crRNA, which has not been seen in other Acr proteins against the Csy complex. Our structure-guided mutagenesis and biochemistry experiments further support the anti-CRISPR mechanisms of these Acr proteins. Our findings support the convergent consequence of inhibiting degradation of invading DNA by these Acr proteins, albeit with different modes of interactions with the type I-F CRISPR-Cas system.
View details for DOI 10.1073/pnas.1922638117
View details for PubMedID 32170016
- Cryo-EM structures of Helicobacter pylori vacuolating cytotoxin A oligomeric assemblies at near-atomic resolution PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2019; 116 (14): 6800–6805
Cryo-EM structure of a 40 kDa SAM-IV riboswitch RNA at 3.7 Å resolution.
2019; 10 (1): 5511
Specimens below 50 kDa have generally been considered too small to be analyzed by single-particle cryo-electron microscopy (cryo-EM). The high flexibility of pure RNAs makes it difficult to obtain high-resolution structures by cryo-EM. In bacteria, riboswitches regulate sulfur metabolism through binding to the S-adenosylmethionine (SAM) ligand and offer compelling targets for new antibiotics. SAM-I, SAM-I/IV, and SAM-IV are the three most commonly found SAM riboswitches, but the structure of SAM-IV is still unknown. Here, we report the structures of apo and SAM-bound SAM-IV riboswitches (119-nt, ~40 kDa) to 3.7 Å and 4.1 Å resolution, respectively, using cryo-EM. The structures illustrate homologies in the ligand-binding core but distinct peripheral tertiary contacts in SAM-IV compared to SAM-I and SAM-I/IV. Our results demonstrate the feasibility of resolving small RNAs with enough detail to enable detection of their ligand-binding pockets and suggest that cryo-EM could play a role in structure-assisted drug design for RNA.
View details for DOI 10.1038/s41467-019-13494-7
View details for PubMedID 31796736
A Single Immunization with Spike-Functionalized Ferritin Vaccines Elicits Neutralizing Antibody Responses against SARS-CoV-2 in Mice.
ACS central science
2021; 7 (1): 183–99
The development of a safe and effective SARS-CoV-2 vaccine is a public health priority. We designed subunit vaccine candidates using self-assembling ferritin nanoparticles displaying one of two multimerized SARS-CoV-2 spikes: full-length ectodomain (S-Fer) or a C-terminal 70 amino-acid deletion (SDeltaC-Fer). Ferritin is an attractive nanoparticle platform for production of vaccines, and ferritin-based vaccines have been investigated in humans in two separate clinical trials. We confirmed proper folding and antigenicity of spike on the surface of ferritin by cryo-EM and binding to conformation-specific monoclonal antibodies. After a single immunization of mice with either of the two spike ferritin particles, a lentiviral SARS-CoV-2 pseudovirus assay revealed mean neutralizing antibody titers at least 2-fold greater than those in convalescent plasma from COVID-19 patients. Additionally, a single dose of SDeltaC-Fer elicited significantly higher neutralizing responses as compared to immunization with the spike receptor binding domain (RBD) monomer or spike ectodomain trimer alone. After a second dose, mice immunized with SDeltaC-Fer exhibited higher neutralizing titers than all other groups. Taken together, these results demonstrate that multivalent presentation of SARS-CoV-2 spike on ferritin can notably enhance elicitation of neutralizing antibodies, thus constituting a viable strategy for single-dose vaccination against COVID-19.
View details for DOI 10.1021/acscentsci.0c01405
View details for PubMedID 33527087
- Resolving individualatoms of protein complex by cryo-electron microscopy. Cell research 2020
- 3D RNA nanocage for encapsulation and shielding of hydrophobic biomolecules to improve the in vivo biodistribution NANO RESEARCH 2020
Cryo-EM Structures of Human Drosha and DGCR8 in Complex with Primary MicroRNA.
Metazoan microRNAs require specific maturation steps initiated by Microprocessor, comprising Drosha and DGCR8. Lack of structural information for the assembled complex has hindered an understanding of how Microprocessor recognizes primary microRNA transcripts (pri-miRNAs). Here we present a cryoelectron microscopy structure of human Microprocessor with a pri-miRNA docked in the active site, poised for cleavage. The basal junction is recognized by a four-way intramolecular junction in Drosha, triggered by the Belt and Wedge regions that clamp over the ssRNA. The belt is important for efficiency and accuracy of pri-miRNA processing. Two dsRBDs form a molecular ruler to measure the stem length between the two dsRNA-ssRNA junctions. The specific organization of the dsRBDs near the apical junction is independent of Drosha core domains, as observed in a second structure in the partially docked state. Collectively, we derive a molecular model to explain how Microprocessor recognizes a pri-miRNA and accurately identifies the cleavage site.
View details for DOI 10.1016/j.molcel.2020.02.016
View details for PubMedID 32220646
Structure of the G protein chaperone and guanine nucleotide exchange factor Ric-8A bound to Galphai1.
2020; 11 (1): 1077
Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Galpha) and serves as an essential Galpha chaperone. Mechanisms by which Ric-8A catalyzes these activities, which are stimulated by Casein Kinase II phosphorylation, are unknown. We report the structure of the nanobody-stabilized complex of nucleotide-free Galpha bound to phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. The mechanism of Ric-8A GEF activity differs considerably from that employed by G protein-coupled receptors at the plasma membrane. Ric-8A engages a specific conformation of Galpha at multiple interfaces to form a complex that is stabilized by phosphorylation within a Ric-8A segment that connects two Galpha binding sites. The C-terminus of Galpha is ejected from its beta sheet core, thereby dismantling the GDP binding site. Ric-8A binds to the exposed Galpha beta sheet and switch II to stabilize the nucleotide-free state of Galpha.
View details for DOI 10.1038/s41467-020-14943-4
View details for PubMedID 32103024
Ultra-thermostable RNA nanoparticles for solubilizing and high-yield loading of paclitaxel for breast cancer therapy.
2020; 11 (1): 972
Paclitaxel is widely used in cancer treatments, but poor water-solubility and toxicity raise serious concerns. Here we report an RNA four-way junction nanoparticle with ultra-thermodynamic stability to solubilize and load paclitaxel for targeted cancer therapy. Each RNA nanoparticle covalently loads twenty-four paclitaxel molecules as aprodrug. The RNA-paclitaxel complex is structurally rigid and stable, demonstrated by the sub-nanometer resolution imaging of cryo-EM. Using RNA nanoparticles as carriers increases the water-solubility of paclitaxel by 32,000-fold. Intravenous injections of RNA-paclitaxel nanoparticles with specific cancer-targeting ligand dramatically inhibit breast cancer growth, with nearly undetectable toxicity and immune responses in mice. No fatalities are observed at a paclitaxel dose equal to the reported LD50. The use of ultra-thermostable RNA nanoparticles to deliver chemical prodrugs addresses issues with RNA unfolding and nanoparticle dissociation after high-density drug loading. This finding provides a stable nano-platform for chemo-drug delivery as well as an efficient method to solubilize hydrophobic drugs.
View details for DOI 10.1038/s41467-020-14780-5
View details for PubMedID 32080195
Measurement of atom resolvability in cryo-EM maps with Q-scores.
Cryogenic electron microscopy (cryo-EM) maps are now at the point where resolvability of individual atoms can be achieved. However, resolvability is not necessarily uniform throughout the map. We introduce a quantitative parameter to characterize the resolvability of individual atoms in cryo-EM maps, the map Q-score. Q-scores can be calculated for atoms in proteins, nucleic acids, water, ligands and other solvent atoms, using models fitted to or derived from cryo-EM maps. Q-scores can also be averaged to represent larger features such as entire residues and nucleotides. Averaged over entire models, Q-scores correlate very well with the estimated resolution of cryo-EM maps for both protein and RNA. Assuming the models they are calculated from are well fitted to the map, Q-scores can be used as a measure of resolvability in cryo-EM maps at various scales, from entire macromolecules down to individual atoms. Q-score analysis of multiple cryo-EM maps of the same proteins derived from different laboratories confirms the reproducibility of structural features from side chains down to water and ion atoms.
View details for DOI 10.1038/s41592-020-0731-1
View details for PubMedID 32042190
Accelerated cryo-EM-guided determination of three-dimensional RNA-only structures.
2020; 17 (7): 699–707
The discovery and design of biologically important RNA molecules is outpacing three-dimensional structural characterization. Here, we demonstrate that cryo-electron microscopy can routinely resolve maps of RNA-only systems and that these maps enable subnanometer-resolution coordinate estimation when complemented with multidimensional chemical mapping and Rosetta DRRAFTER computational modeling. This hybrid 'Ribosolve' pipeline detects and falsifies homologies and conformational rearrangements in 11 previously unknown 119- to 338-nucleotide protein-free RNA structures: full-length Tetrahymena ribozyme, hc16 ligase with and without substrate, full-length Vibrio cholerae and Fusobacterium nucleatum glycine riboswitch aptamers with and without glycine, Mycobacterium SAM-IV riboswitch with and without S-adenosylmethionine, and the computer-designed ATP-TTR-3 aptamer with and without AMP. Simulation benchmarks, blind challenges, compensatory mutagenesis, cross-RNA homologies and internal controls demonstrate that Ribosolve can accurately resolve the global architectures of RNA molecules but does not resolve atomic details. These tests offer guidelines for making inferences in future RNA structural studies with similarly accelerated throughput.
View details for DOI 10.1038/s41592-020-0878-9
View details for PubMedID 32616928
A single immunization with spike-functionalized ferritin vaccines elicits neutralizing antibody responses against SARS-CoV-2 in mice.
bioRxiv : the preprint server for biology
Development of a safe and effective SARS-CoV-2 vaccine is a public health priority. We designed subunit vaccine candidates using self-assembling ferritin nanoparticles displaying one of two multimerized SARS-CoV-2 spikes: full-length ectodomain (S-Fer) or a C-terminal 70 amino-acid deletion (SΔC-Fer). Ferritin is an attractive nanoparticle platform for production of vaccines and ferritin-based vaccines have been investigated in humans in two separate clinical trials. We confirmed proper folding and antigenicity of spike on the surface of ferritin by cryo-EM and binding to conformation-specific monoclonal antibodies. After a single immunization of mice with either of the two spike ferritin particles, a lentiviral SARS-CoV-2 pseudovirus assay revealed mean neutralizing antibody titers at least 2-fold greater than those in convalescent plasma from COVID-19 patients. Additionally, a single dose of SΔC-Fer elicited significantly higher neutralizing responses as compared to immunization with the spike receptor binding domain (RBD) monomer or spike ectodomain trimer alone. After a second dose, mice immunized with SΔC-Fer exhibited higher neutralizing titers than all other groups. Taken together, these results demonstrate that multivalent presentation of SARS-CoV-2 spike on ferritin can notably enhance elicitation of neutralizing antibodies, thus constituting a viable strategy for single-dose vaccination against COVID-19.
View details for DOI 10.1101/2020.08.28.272518
View details for PubMedID 32869030
View details for PubMedCentralID PMC7457616
- Coupling of ssRNA cleavage with DNase activity in type III-A CRISPR-Csm revealed by cryo-EM and biochemistry CELL RESEARCH 2019; 29 (4): 305–12
Automated Sequence Design of 3D Polyhedral Wireframe DNA Origami with Honeycomb Edges
2019; 13 (2): 2083–93
3D polyhedral wireframe DNA nanoparticles (DNA-NPs) fabricated using scaffolded DNA origami offer complete and independent control over NP size, structure, and asymmetric functionalization on the 10-100 nm scale. However, the complex DNA sequence design needed for the synthesis of these versatile DNA-NPs has limited their widespread use to date. While the automated sequence design algorithms DAEDALUS and vHelix-BSCOR apply to DNA-NPs synthesized using either uniformly dual or hybrid single-dual duplex edges, respectively, these DNA-NPs are relatively compliant mechanically and are therefore of limited utility for some applications. Further, these algorithms are incapable of handling DNA-NP edge designs composed of more than two duplexes, which are needed to enhance DNA-NP mechanical stiffness. As an alternative, here we introduce the scaffolded DNA origami sequence design algorithm TALOS, which is a generalized procedure for the fully automated design of wireframe 3D polyhedra composed of edges of any cross section with an even number of duplexes, and apply it to DNA-NPs composed uniformly of single honeycomb edges. We also introduce a multiway vertex design that enables the fabrication of DNA-NPs with arbitrary edge lengths and vertex angles and apply it to synthesize a highly asymmetric origami object. Sequence designs are demonstrated to fold robustly into target DNA-NP shapes with high folding efficiency and structural fidelity that is verified using single particle cryo-electron microscopy and 3D reconstruction. In order to test its generality, we apply TALOS to design an in silico library of over 200 DNA-NPs of distinct symmetries and sizes, and for broad impact, we also provide the software as open source for the generation of custom NP designs.
View details for PubMedID 30605605
TRPC Channels and Glioma
TRANSIENT RECEPTOR POTENTIAL CANONICAL CHANNELS AND BRAIN DISEASES
2017; 976: 157–65
Glioma is the most common type of brain tumors and malignant glioma is extremely lethal, with patients' 5-year survival rate less than 10%. Treatment of gliomas poses remarkable clinical challenges, not only because of their particular localization but also because glioma cells possess several malignant biological features, including highly proliferative, highly invasive, highly angiogenic, and highly metabolic aberrant. All these features make gliomas highly recurrent and drug resistant. Finding new and effective molecular drug targets for glioma is an urgent and critical task for both basic and clinical research. Recent studies have proposed a type of non-voltage-gated calcium channels, namely, canonical transient receptor potential (TRPC) channels, to be newly emerged potential drug targets for glioma. They are heavily involved in the proliferation, migration, invasion, angiogenesis, and metabolism of glioma cells. Abundant evidence from both cell models and preclinical mouse models has demonstrated that inhibition of TRPC channels shows promising anti-glioma effect. In this chapter, we will give a comprehensive review on the current progress in the studies on TRPC channels and glioma and discuss their potential clinical implication in glioma therapy.
View details for DOI 10.1007/978-94-024-1088-4_14
View details for Web of Science ID 000429061700014
View details for PubMedID 28508321
Crucial role of TRPC6 in maintaining the stability of HIF-1 alpha in glioma cells under hypoxia
JOURNAL OF CELL SCIENCE
2015; 128 (17): 3317–29
Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor responsible for the expression of a broad range of genes that facilitate acclimatization to hypoxia. Its stability is predominantly controlled by rapid hydroxylation of two proline residues in its α-subunit. However, how the rapid hydroxylation of HIF-1α is regulated is not fully understood. Here, we report that transient receptor potential canonical (TRPC) 6 channels control hydroxylation and stability of HIF-1α in human glioma cells under hypoxia. TRPC6 was rapidly activated by IGF-1R-PLCγ-IP3R pathway upon hypoxia. Inhibition of TRPC6 enhanced the levels of α-ketoglutarate and promoted hydroxylation of HIF-1α to suppress HIF-1α accumulation without affecting its transcription or translation. Dimethyloxalylglycine N-(methoxyoxoacetyl)-glycine methyl ester (DMOG), an analog of α-ketoglutarate, reversed the inhibition of HIF-1α accumulation. Moreover, TRPC6 regulated GLUT1 (also known as SLC2A1) expression in a manner that was dependent on HIF-1α accumulation to affect glucose uptake during hypoxia. Our results suggest that TRPC6 regulates metabolism to affect HIF-1α stability and consequent glucose metabolism in human glioma cells under hypoxia.
View details for DOI 10.1242/jcs.173161
View details for Web of Science ID 000360715800014
View details for PubMedID 26187851