9 Sep 2022

Intermediates in SARS-CoV-2 spike-mediated cell entry (Science Advances)

SARS-CoV-2 cell entry is completed after viral spike (S) protein-mediated membrane fusion between viral and host cell membranes. Stable prefusion and postfusion S structures have been resolved by cryo-electron microscopy and cryo-electron tomography, but the refolding intermediates on the fusion pathway are transient and have not been examined. We used an antiviral lipopeptide entry inhibitor to arrest S protein refolding and thereby capture intermediates as S proteins interact with hACE2 and fusion-activating proteases on cell-derived target membranes. Cryo-electron tomography imaged both extended and partially folded intermediate states of S2, as well as a novel late-stage conformation on the pathway to membrane fusion. The intermediates now identified in this dynamic S protein-directed fusion provide mechanistic insights that may guide the design of CoV entry inhibitors.

9 Sep 2022

A public antibody class recognizes an S2 epitope exposed on open conformations of SARS-CoV-2 spike (Nature Communications)

Delineating the origins and properties of antibodies elicited by SARS-CoV-2 infection and vaccination is critical for understanding their benefits and potential shortcomings. Therefore, we investigate the SARS-CoV-2 spike (S)-reactive B cell repertoire in unexposed individuals by flow cytometry and single-cell sequencing. We show that similar to 82% of SARS-CoV-2 S-reactive B cells harbor a naive phenotype, which represents an unusually high fraction of total human naive B cells (similar to 0.1%). Approximately 10% of these naive S-reactive B cells share an IGHV1-69/IGKV3-11 B cell receptor pairing, an enrichment of 18-fold compared to the complete naive repertoire. Following SARS-CoV-2 infection, we report an average 37-fold enrichment of IGHV1-69/IGKV3-11 B cell receptor pairing in the S-reactive memory B cells compared to the unselected memory repertoire. This class of B cells targets a previously undefined non-neutralizing epitope on the S2 subunit that becomes exposed on S proteins used in approved vaccines when they transition away from the native pre-fusion state because of instability. These findings can help guide the improvement of SARS-CoV-2 vaccines.

To fully understand the potential shortcomings of SARS-CoV-2 vaccination, it is necessary to delineate the properties of the antibodies elicited, during immunization, and also infection. Through investigation of the SARS-CoV-2 spike-reactive B cell repertoire, authors identify following infection, a subset of B cells enriched and almost exclusively target a non-neutralizing S2 epitope present in aberrant forms.

9 Sep 2022

Structural insights of a highly potent pan-neutralizing SARS-CoV-2 human monoclonal antibody (PNAS)

As the coronavirus disease 2019 (COVID-19) pandemic continues, there is a strong need for highly potent monoclonal antibodies (mAbs) that are resistant against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VoCs). Here, we evaluate the potency of the previously described mAb J08 against these variants using cell-based assays and delve into the molecular details of the binding interaction using cry-oelectron microscopy (cryo-EM) and X-ray crystallography. We show that mAb J08 has low nanomolar affinity against most VoCs and binds high on the receptor binding domain (RBD) ridge, away from many VoC mutations. These findings further validate the phase II/III human clinical trial underway using mAb J08 as a monoclonal therapy.

10 Oct 2023

cGLRs are a diverse family of pattern recognition receptors in innate immunity (Cell)

Cyclic GMP-AMP synthase (cGAS) is an enzyme in human cells that controls an immune response to cytosolic DNA. Upon binding DNA, cGAS synthesizes a nucleotide signal 2′3′-cGAMP that activates STING-dependent downstream immunity. Here, we discover that cGAS-like receptors (cGLRs) constitute a major family of pattern recognition receptors in innate immunity. Building on recent analysis in Drosophila, we identify >3,000 cGLRs present in nearly all metazoan phyla. A forward biochemical screening of 150 animal cGLRs reveals a conserved mechanism of signaling including response to dsDNA and dsRNA ligands and synthesis of isomers of the nucleotide signals cGAMP, c-UMP-AMP, and c-di-AMP. Combining structural biology and in vivo analysis in coral and oyster animals, we explain how the synthesis of distinct nucleotide signals enables cells to control discrete cGLR-STING signaling pathways. Our results reveal cGLRs as a widespread family of pattern recognition receptors and establish molecular rules that govern nucleotide signaling in animal immunity.

10 Oct 2023

Structural basis of γ chain family receptor sharing at the membrane level (Science)

Common γ chain (γc) cytokine receptors, including interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15, and IL-21 receptors, are activated upon engagement with a common γc receptor (CD132) by concomitant binding of their ectodomains to an interleukin. In this work, we find that direct interactions between the transmembrane domains (TMDs) of both the γc and the interleukin receptors (ILRs) are also required for receptor activation. Moreover, the same γc TMD can specifically recognize multiple ILR TMDs of diverse sequences within the family. Heterodimer structures of γc TMD bound to IL-7 and IL-9 receptor TMDs—determined in a lipid bilayer–like environment by nuclear magnetic resonance spectroscopy—reveal a conserved knob-into-hole mechanism of recognition that mediates receptor sharing within the membrane. Thus, signaling in the γc receptor family requires specific heterotypic interactions of the TMDs.

10 Oct 2023

After AlphaFold: protein-folding contest seeks next big breakthrough (Nature)

Two years after DeepMind’s revolutionary AI swept a competition for predicting protein structures, researchers are building on AlphaFold’s success. “In some sense, the problem is solved,” computational biologist John Moult declared in late 2020. The London-based company DeepMind had just swept a biennial contest co-founded by Moult that tests teams’ abilities to predict protein structures — one of biology’s grandest challenges — with its revolutionary artificial-intelligence (AI) tool AlphaFold. Two years later, Moult’s competition, the Critical Assessment of Structure Prediction (CASP), is still walking in AlphaFold’s long shadow. Results from this year’s edition (CASP15) — which were unveiled over the weekend at a conference in Antalya, Turkey — show that the most successful approaches to predicting protein structures from their amino-acid sequences incorporated AlphaFold, which relies on an AI approach called deep learning. “Everyone is using AlphaFold,” says Yang Zhang, a computational biologist at the University of Michigan in Ann Arbor. Yet AlphaFold’s progress has opened the floodgates for new challenges in protein-structure prediction — some included in this year’s CASP — that might require new approaches and more time to fully tackle. “The low-hanging fruit has been picked,” says Mohammed AlQuraishi, a computational biologist at Columbia University in New York City. “Some of the next problems are going to be harder.”

29 Aug 2023

Cryo-EM structure of coagulation factor V short (Blood)

Coagulation factor V (fV) is the precursor of activated fV (fVa), an essential component of the prothrombinase complex required for the rapid activation of prothrombin in the penultimate step of the coagulation cascade. In addition, fV regulates the tissue factor pathway inhibitor α (TFPIα) and protein C pathways that inhibit the coagulation response. A recent cryogenic electron microscopy (cryo-EM) structure of fV has revealed the architecture of its A1-A2-B-A3-C1-C2 assembly but left the mechanism that keeps fV in its inactive state unresolved because of an intrinsic disorder in the B domain. A splice variant of fV, fV short, carries a large deletion of the B domain that produces constitutive fVa-like activity and unmasks epitopes for the binding of TFPIα. The cryo-EM structure of fV short was solved at 3.2 Å resolution and revealed the arrangement of the entire A1-A2-B-A3-C1-C2 assembly. The shorter B domain stretches across the entire width of the protein, making contacts with the A1, A2, and A3 domains but suspended over the C1 and C2 domains. In the portion distal to the splice site, several hydrophobic clusters and acidic residues provide a potential binding site for the basic C-terminal end of TFPIα. In fV, these epitopes may bind intramolecularly to the basic region of the B domain. The cryo-EM structure reported in this study advances our understanding of the mechanism that keeps fV in its inactive state, provides new targets for mutagenesis and facilitates future structural analysis of fV short in complex with TFPIα, protein S, and fXa.

29 Aug 2023

Principles of human pre-60S biogenesis (Science)

Human ribosome biogenesis is a highly regulated process involving a vast ensemble of assembly factors that orchestrate the formation of both ribosomal subunits. The mechanisms underlying the early assembly of the human large ribosomal subunit (pre-60S) remain elusive. To provide insights into human ribosome assembly, Vanden Broeck and Klinge used a combination of genome editing, cryo–electron microscopy, and functional studies to reveal the structures of human pre-60S particles as they matured in the nucleolus and nucleus. This collection of structures highlights how assembly factors ensure the unidirectional maturation of human large ribosomal subunit precursors. The study set out to elucidate the early stages of human pre-60S assembly at a mechanistic level. To this end, the authors have combined human genome editing and biochemistry to permeabilize human nucleoli and nuclei to isolate intact endogenous pre-60Sassembly intermediates for subsequent structural characterization by cryo–electron microscopy (cryo-EM). To functionally study rRNA processing, they developed an in vivo recombinant ribosome assembly assay using an engineered human rDNA locus to investigate whether rRNA elements within ITS2 and the 28S rRNA are required for large ribosomal subunit biogenesis.

29 Aug 2023

TDP-43 forms amyloid filaments with a distinct fold in type A FTLD-TDP (Nature)

The abnormal assembly of TAR DNA-binding protein 43 (TDP-43) in neuronal and glial cells characterizes nearly all cases of amyotrophic lateral sclerosis (ALS) and around half of cases of frontotemporal lobar degeneration (FTLD). A causal role for TDP-43 assembly in neurodegeneration is evidenced by dominantly inherited missense mutations in TARDBP, the gene encoding TDP-43, that promote assembly and give rise to ALS and FTLD. At least four types (A–D) of FTLD with TDP-43 pathology (FTLD-TDP) are defined by distinct brain distributions of assembled TDP-43 and are associated with different clinical presentations of frontotemporal dementia. We previously showed, using cryo-electron microscopy, that TDP-43 assembles into amyloid filaments in ALS and type B FTLD-TDP. However, the structures of assembled TDP-43 in FTLD without ALS remained unknown. Here we report the cryo-electron microscopy structures of assembled TDP-43 from the brains of three individuals with the most common type of FTLD-TDP, type A. TDP-43 formed amyloid filaments with a new fold that was the same across individuals, indicating that this fold may characterize type A FTLD-TDP. The fold resembles a chevron badge and is unlike the double-spiral-shaped fold of ALS and type B FTLD-TDP, establishing that distinct filament folds of TDP-43 characterize different neurodegenerative conditions. The structures, in combination with mass spectrometry, led to the identification of two new post-translational modifications of assembled TDP-43, citrullination and monomethylation of R293, and indicate that they may facilitate filament formation and observed structural variation in individual filaments. The structures of TDP-43 filaments from type A FTLD-TDP will guide mechanistic studies of TDP-43 assembly, as well as the development of diagnostic and therapeutic compounds for TDP-43 proteinopathies.

18 Aug 2023

Discovery of VH domains that allosterically inhibit ENPP1 (Nature Chemical Biology)

The origin of viruses remains an open question. While lack of detectable sequence similarity hampers the analysis of distantly related viruses, structural biology investigations of conserved capsid protein structures facilitate the study of distant evolutionary relationships. Here we characterize the lipidcontaining ssDNA temperate bacteriophage Phi CjT23, which infects Flavobacterium sp. (Bacteroidetes). We report Phi CjT23-like sequences in the genome of strains belonging to several Flavobacterium species. The virion structure determined by cryogenic electronmicroscopy reveals similarities to members of the viral kingdom Bamfordvirae that currently consists solely of dsDNA viruses with a major capsid protein composed of two upright beta-sandwiches. The minimalistic structure of Phi CjT23 suggests that this phage serves as a model for the last common ancestor between ssDNA and dsDNA viruses in the Bamfordvirae. Both Phi CjT23 and the related phage FLiP infect Flavobacterium species found in several environments, suggesting that these types of viruses have a global distribution and a shared evolutionary origin. Detailed comparisons to related, more complex viruses not only expand our knowledge about this group of viruses but also provide a rare glimpse into early virus evolution.

18 Aug 2023

Structural basis for specific RNA recognition by the alternative splicing factor RBM5 (Nature Communications)

The RNA-binding motif protein RBM5 belongs to a family of multi-domain RNA binding proteins that regulate alternative splicing of genes important for apoptosis and cell proliferation and have been implicated in cancer. RBM5 harbors structural modules for RNA recognition, such as RRM domains and a Zn finger, and protein-protein interactions such as an OCRE domain. Here, we characterize binding of the RBM5 RRM1-ZnF1-RRM2 domains to cis-regulatory RNA elements. A structure of the RRM1-ZnF1 region in complex with RNA shows how the tandem domains cooperate to sandwich target RNA and specifically recognize a GG dinucleotide in a non-canonical fashion. While the RRM1-ZnF1 domains act as a single structural module, RRM2 is connected by a flexible linker and tumbles independently. However, all three domains participate in RNA binding and adopt a closed architecture upon RNA binding. Our data highlight how cooperativity and conformational modularity of multiple RNA binding domains enable the recognition of distinct RNA motifs, thereby contributing to the regulation of alternative splicing. Remarkably, we observe surprising differences in coupling of the RNA binding domains between the closely related homologs RBM5 and RBM10. The RNA binding protein RBM5 regulates alternative splicing of genes implicated in cancer. Here the authors show structural mechanisms how multiple RNA binding domains of RBM5 cooperate to recognize specific target RNA sequences.