9 Sep 2022

SARS-CoV-2 variants of concern: spike protein mutational analysis and epitope for broad neutralization (Nature Communications)

Mutations in the spike glycoproteins of SARS-CoV-2 variants of concern have independently been shown to enhance aspects of spike protein fitness. Here, we describe an antibody fragment (V-H ab6) that neutralizes all major variants including the recently emerged BA.1 and BA.2 Omicron subvariants, with a unique mode of binding revealed by cryo-EM studies. Further, we provide a comparative analysis of the mutational effects within previously emerged variant spikes and identify the structural role of mutations within the NTD and RBD in evading antibody neutralization. Our analysis shows that the highly mutated Gamma N-terminal domain exhibits considerable structural rearrangements, partially explaining its decreased neutralization by convalescent sera. Our results provide mechanistic insights into the structural, functional, and antigenic consequences of SARS-CoV-2 spike mutations and highlight a spike protein vulnerability that may be exploited to achieve broad protection against circulating variants.

SARS-CoV-2 variants have accumulated multiple defining mutations within their spike glycoproteins. Here, the authors report a structural basis for broad neutralization of several variants by a heavy chain antibody fragment and provide a mutational analysis focusing on antibody evasion, receptor engagement, and spike protein structure.

9 Sep 2022

Omicron SARS-CoV-2 mutations stabilize spike up-RBD conformation and lead to a non-RBM-binding monoclonal antibody escape (Nature Communications)

Omicron SARS-CoV-2 is rapidly spreading worldwide. To delineate the impact of emerging mutations on spike's properties, we performed systematic structural analyses on apo Omicron spike and its complexes with human ACE2 or S309 neutralizing antibody (NAb) by cryo-EM. The Omicron spike preferentially adopts the one-RBD-up conformation both before and after ACE2 binding, which is in sharp contrast to the orchestrated conformational changes to create more up-RBDs upon ACE2 binding as observed in the prototype and other four variants of concern (VOCs). Furthermore, we found that S371L, S373P and S375F substitutions enhance the stability of the one-RBD-up conformation to prevent exposing more up-RBDs triggered by ACE2 binding. The increased stability of the one-RBD-up conformation restricts the accessibility of S304 NAb, which targets a cryptic epitope in the closed conformation, thus facilitating the immune evasion by Omicron. These results expand our understanding of Omicron spike's conformation, receptor binding and antibody evasion mechanism.

The SARS-CoV-2 Omicron variant spreads rapidly. Here the authors show that Omicron S preferentially adopts the one-RBD-up conformation, which leads to a non-RBM-binding monoclonal antibody escape. Mutagenesis reveals that S371L, S373P and S375F substitutions enhance the conformational stability.

9 Sep 2022

Broadly neutralizing antibodies target the coronavirus fusion peptide (Science)

The potential for future coronavirus outbreaks highlights the need to broadly target this group of pathogens. We used an epitope-agnostic approach to identify six monoclonal antibodies that bind to spike proteins from all seven human-infecting coronaviruses. All six antibodies target the conserved fusion peptide region adjacent to the S2' cleavage site. COV44-62 and COV44-79 broadly neutralize alpha- and betacoronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants BA.2 and BA.4/5, albeit with lower potency than receptor binding domain-specific antibodies. In crystal structures of COV44-62 and COV44-79 antigen-binding fragments with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine residue at the S2' cleavage site. COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings highlight the fusion peptide as a candidate epitope for next-generation coronavirus vaccine development.

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.

8 Aug 2023

Exploration of novel αβ-protein folds through de novo design (Nature Structural & Molecular Biology)

A fundamental question in protein evolution is whether nature has exhaustively sampled nearly all possible protein folds throughout evolution, or whether a large fraction of the possible folds remains unexplored. To address this question, we defined a set of rules for β-sheet topology to predict novel αβ-folds and carried out a systematic de novo protein design exploration of the novel αβ-folds predicted by the rules. The designs for all eight of the predicted novel αβ-folds with a four-stranded β-sheet, including a knot-forming one, folded into structures close to the design models. Further, the rules predicted more than 10,000 novel αβ-folds with five- to eight-stranded β-sheets; this number far exceeds the number of αβ-folds observed in nature so far. This result suggests that a vast number of αβ-folds are possible, but have not emerged or have become extinct due to evolutionary bias.

8 Aug 2023

Orchestrating nucleic acid-protein interactions at chromosome ends: telomerase mechanisms come into focus (Nature Structural & Molecular Biology)

Telomerase is a special reverse transcriptase ribonucleoprotein dedicated to the synthesis of telomere repeats that protect chromosome ends. Among reverse transcriptases, telomerase is unique in using a stably associated RNA with an embedded template to synthesize a specified sequence. Moreover, it is capable of iteratively copying the same template region (repeat addition processivity) through multiple rounds of RNA–DNA unpairing and reannealing, that is, the translocation reaction. Biochemical analyses of telomerase over the past 3 decades in protozoa, fungi and mammals have identified structural elements that underpin telomerase mechanisms and have led to models that account for the special attributes of telomerase. Notably, these findings and models can now be interpreted and adjudicated through recent cryo-EM structures of Tetrahymena and human telomerase holoenzyme complexes in association with substrates and regulatory proteins. Collectively, these structures reveal the intricate protein–nucleic acid interactions that potentiate telomerase’s unique translocation reaction and clarify how this enzyme reconfigures the basic reverse transcriptase scaffold to craft a polymerase dedicated to the synthesis of telomere DNA. Among the many new insights is the resolution of the telomerase ‘anchor site’ proposed more than 3 decades ago. The structures also highlight the nearly universal conservation of a protein–protein interface between an oligonucleotide/oligosaccharide-binding (OB)-fold regulatory protein and the telomerase catalytic subunit, which enables spatial and temporal regulation of telomerase function in vivo. In this Review, we discuss key features of the structures in combination with relevant functional analyses. We also examine conserved and divergent aspects of telomerase mechanisms as gleaned from studies in different model organisms.