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Highlights of Coronavirus Structural Studies

5 Jan

The atomic portrait of SARS-CoV-2 as captured by cryo-electron microscopy (Journal of Cellular and Molecular Medicine)

Transmission electron microscopy has historically been indispensable for virology research, as it offers unique insight into virus function. In the past decade, as cryo-electron microscopy (cryo-EM) has matured and become more accessible, we have been able to peer into the structure of viruses at the atomic level and understand how they interact with the host cell, with drugs or with antibodies. Perhaps, there was no time in recent history where cryo-EM was more needed, as SARS-CoV-2 has spread around the globe, causing millions of deaths and almost unquantifiable economic devastation. In this concise review, we aim to mark the most important contributions of cryo-EM to understanding the structure and function of SARS-CoV-2 proteins, from surface spikes to the virus core and from virus-receptor interactions to antibody binding.

5 Jan

Membrane fusion and immune evasion by the spike protein of SARS-CoV-2 Delta variant (Science)

The Delta variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has outcompeted previously prevalent variants and become a dominant strain worldwide. We report the structure, function, and antigenicity of its full-length spike (S) trimer as well as those of the Gamma and Kappa variants, and compare their characteristics with the G614, Alpha, and Beta variants. Delta S can fuse membranes more efficiently at low levels of cellular receptor angiotensin converting enzyme 2 (ACE2), and its pseudotyped viruses infect target cells substantially faster than the other five variants, possibly accounting for its heightened transmissibility. Each variant shows different rearrangement of the antigenic surface of the amino-terminal domain of the S protein but only makes produces changes in the receptor binding domain (RBD), making the RBD a better target for therapeutic antibodies.

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Research Highlights

the best of science obtained using CIISB Core Facilities

EMBO Journal 2022

Cryo-EM structures of CspA27 inside the exit tunnel

  1. Structure and nascent chain contacts in the exit tunnel for CspA27-1.
  2. Structure and nascent chain contacts in the exit tunnel for CspA27-2.
  3. Structure and nascent chain contacts in the exit tunnel for CspA27-3.

Marina V. Rodnina Research Group

Significance

Cellular proteins begin to fold as they emerge from the ribosome. The folding landscape of nascent chains is not only shaped by their amino acid sequence but also by the interactions with the ribosome. Here, we combine biophysical methods with cryo-EM structure determination to show that folding of a β-barrel protein begins with formation of a dynamic α-helix inside the ribosome. As the growing peptide reaches the end of the tunnel, the N-terminal part of the nascent chain refolds to a β-hairpin structure that remains dynamic until its release from the ribosome. Contacts with the ribosome and structure of the peptidyl transferase center depend on nascent chain conformation. These results indicate that proteins may start out as α-helices inside the tunnel and switch into their native folds only as they emerge from the ribosome. Moreover, the correlation of nascent chain conformations with reorientation of key residues of the ribosomal peptidyl-transferase center suggest that protein folding could modulate ribosome activity.

Agirrezabala, X., Samatova, E., Macher, M., Liutkute, M., Maiti, M., Gil-Carton, D., Novacek, J., Valle. M., and Rodnina, M.V.: EMBO J. (2022) e109175, https://doi.org/10.15252/embj.2021109175

 

ACS Nano 2021

Snapshots from simulations representing the release pathways. The rapid pathway category (R1–R3) was divided into (R1) burst genome release, where capsids disintegrate into fragments; (R2) rupture genome release, where capsids split open (most often into two halves); and (R3) bloom genome release, where capsids open wide in one hemisphere without breaking the other. In the rupture and bloom pathways, a majority of the capsid reassembles after the genome release. Occasionally, pentamers of capsid proteins may be detached. The subcategory leaky release (L) started with a slow release and ended with a rapid release. The slow pathway (S1–S3) category was divided into (S1) release through a pore on a twofold axis, (S2) release through a pore on a threefold axis, and (S3) release through multiple pores. Note that slow release was only observed for the noncompact genome. All release examples are shown with the noncompact genome. Color coding: Beads forming the body of the capsid are orange on the outside and purple on the inside of the capsid. The genome is represented by blue beads. Red and green beads represent attractive beads between pentamers.

Robert Vácha Research Group

Significance

Virus-like nanoparticles are protein shells similar to wild-type viruses, and both aim to deliver their content into a cell. Unfortunately, the release mechanism of their cargo/ genome remains elusive. Pores on the symmetry axes were proposed to enable the slow release of the viral genome. In contrast, cryo-EM images showed that capsids of nonenveloped RNA viruses can crack open and rapidly release the genome. We combined in vitro cryo-EM observations of the genome release of three viruses with coarse-grained simulations of generic virus-like nanoparticles to investigate the cargo/genome release pathways. Simulations provided details on both slow and rapid release pathways, including the success rates of individual releases. Moreover, the simulated structures from the rapid release pathway were in agreement with the experiment. Slow release occurred when interactions between capsid subunits were long-ranged, and the cargo/genome was noncompact. In contrast, rapid release was preferred when the interaction range was short and/or the cargo/genome was compact. These findings indicate a design strategy of virus-like nanoparticles for drug delivery.

 

Sukeník, L., Mukhamedova, L., Procházková, M., Škubník, K., Plevka, P., and Vácha, R.:

Cargo Release from Nonenveloped Viruses and Virus-like Nanoparticles: Capsid Rupture or Pore Formation, ABC Nano 2021, 15, 12, 19233–19243, https://doi.org/10.1021/acsnano.1c04814

 

More publications Research Highlights archive

Reader’s Corner

literature to read, science to follow

In this section, a distinct selection of six highly stimulating research publications and reviews published during past 6 months is presented. It is our hope that links to exciting science, which deserves attention of the structural biology community, will help you to locate gems in the steadily expanding jungle of scientific literature. You are welcome to point out to any paper you found interesting by sending a link or citation to readerscorner@ciisb.org. The section is being updated regularly.

 

6 Jan

Time-resolved structural analysis of an RNA-cleaving DNA catalyst (Nature)

The 10–23 DNAzyme is one of the most prominent catalytically active DNA sequences. Its ability to cleave a wide range of RNA targets with high selectivity entails a substantial therapeutic and biotechnological potential. However, the high expectations have not yet been met, a fact that coincides with the lack of high-resolution and time-resolved information about its mode of action. Here we provide high-resolution NMR characterization of all apparent states of the prototypic 10–23 DNAzyme and present a comprehensive survey of the kinetics and dynamics of its catalytic function. The determined structure and identified metal-ion-binding sites of the precatalytic DNAzyme–RNA complex reveal that the basis of the DNA-mediated catalysis is an interplay among three factors: an unexpected, yet exciting molecular architecture; distinct conformational plasticity; and dynamic modulation by metal ions. We further identify previously hidden rate-limiting transient intermediate states in the DNA-mediated catalytic process via real-time NMR measurements. Using a rationally selected single-atom replacement, we could considerably enhance the performance of the DNAzyme, demonstrating that the acquired knowledge of the molecular structure, its plasticity and the occurrence of long-lived intermediate states constitutes a valuable starting point for the rational design of next-generation DNAzymes.

6 Jan

De novo protein design by deep network hallucination (Nature)

There has been considerable recent progress in protein structure prediction using deep neural networks to predict inter-residue distances from amino acid sequences. Here we investigate whether the information captured by such networks is sufficiently rich to generate new folded proteins with sequences unrelated to those of the naturally occurring proteins used in training the models. We generate random amino acid sequences, and input them into the trRosetta structure prediction network to predict starting residue–residue distance maps, which, as expected, are quite featureless. We then carry out Monte Carlo sampling in amino acid sequence space, optimizing the contrast (Kullback–Leibler divergence) between the inter-residue distance distributions predicted by the network and background distributions averaged over all proteins. Optimization from different random starting points resulted in novel proteins spanning a wide range of sequences and predicted structures. We obtained synthetic genes encoding 129 of the network-‘hallucinated’ sequences, and expressed and purified the proteins in Escherichia coli; 27 of the proteins yielded monodisperse species with circular dichroism spectra consistent with the hallucinated structures. We determined the three-dimensional structures of three of the hallucinated proteins, two by X-ray crystallography and one by NMR, and these closely matched the hallucinated models. Thus, deep networks trained to predict native protein structures from their sequences can be inverted to design new proteins, and such networks and methods should contribute alongside traditional physics-based models to the de novo design of proteins with new functions.

6 Jan

Structure-guided bifunctional molecules hit a DEUBAD-lacking hRpn13 species upregulated in multiple myeloma (Nature Communications)

Proteasome substrate receptor hRpn13 is a promising anti-cancer target. By integrated in silico and biophysical screening, we identified a chemical scaffold that binds hRpn13 with non-covalent interactions that mimic the proteasome and a weak electrophile for Michael addition. hRpn13 Pru domain binds proteasomes and ubiquitin whereas its DEUBAD domain binds deubiquitinating enzyme UCHL5. NMR revealed lead compound XL5 to interdigitate into a hydrophobic pocket created by lateral movement of a Pru β-hairpin with an exposed end for Proteolysis Targeting Chimeras (PROTACs). Implementing XL5-PROTACs as chemical probes identified a DEUBAD-lacking hRpn13 species (hRpn13Pru) present naturally with cell type-dependent abundance. XL5-PROTACs preferentially target hRpn13Pru, causing its ubiquitination. Gene-editing and rescue experiments established hRpn13 requirement for XL5-PROTAC-triggered apoptosis. These data establish hRpn13 as an anti-cancer target for multiple myeloma and introduce an hRpn13-targeting scaffold that can be optimized for preclinical trials against hRpn13Pru-producing cancer types.

5 Jan

Asymmetric drug binding in an ATP-loaded inward-facing state of an ABC transporter (Nature Chemical Biology)

Substrate efflux by ATP-binding cassette (ABC) transporters, which play a major role in multidrug resistance, entails the ATP-powered interconversion between transporter intermediates. Despite recent progress in structure elucidation, a number of intermediates have yet to be visualized and mechanistically interpreted. Here, we combine cryogenic-electron microscopy (cryo-EM), double electron–electron resonance spectroscopy and molecular dynamics simulations to profile a previously unobserved intermediate of BmrCD, a heterodimeric multidrug ABC exporter from Bacillus subtilis. In our cryo-EM structure, ATP-bound BmrCD adopts an inward-facing architecture featuring two molecules of the substrate Hoechst-33342 in a striking asymmetric head-to-tail arrangement. Deletion of the extracellular domain capping the substrate-binding chamber or mutation of Hoechst-coordinating residues abrogates cooperative stimulation of ATP hydrolysis. Together, our findings support a mechanistic role for symmetry mismatch between the nucleotide binding and the transmembrane domains in the conformational cycle of ABC transporters and is of notable importance for rational design of molecules for targeted ABC transporter inhibition.

5 Jan

Structural and functional diversity among agonist-bound states of the GLP-1 receptor (Nature Chemical Biology)

Recent advances in G-protein-coupled receptor (GPCR) structural elucidation have strengthened previous hypotheses that multidimensional signal propagation mediated by these receptors depends, in part, on their conformational mobility; however, the relationship between receptor function and static structures is inherently uncertain. Here, we examine the contribution of peptide agonist conformational plasticity to activation of the glucagon-like peptide 1 receptor (GLP-1R), an important clinical target. We use variants of the peptides GLP-1 and exendin-4 (Ex4) to explore the interplay between helical propensity near the agonist N terminus and the ability to bind to and activate the receptor. Cryo-EM analysis of a complex involving an Ex4 analog, the GLP-1R and Gs heterotrimer revealed two receptor conformers with distinct modes of peptide–receptor engagement. Our functional and structural data, along with molecular dynamics (MD) simulations, suggest that receptor conformational dynamics associated with flexibility of the peptide N-terminal activation domain may be a key determinant of agonist efficacy.

5 Jan

Mechanism of siRNA production by a plant Dicer-RNA complex in dicing-competent conformation (Science)

In eukaryotes, small RNAs (sRNAs) play critical roles in multiple biological processes. Dicer endonucleases are a central part of sRNA biogenesis. In plants, DICER-LIKE PROTEIN 3 (DCL3) produces 24-nucleotide (nt) small interfering RNAs (siRNAs) that determine the specificity of the RNA-directed DNA methylation pathway. Here, we determined the structure of a DCL3–pre-siRNA complex in an active dicing-competent state. The 5′-phosphorylated A1 of the guide strand and the 1-nt 3′ overhang of the complementary strand are specifically recognized by a positively charged pocket and an aromatic cap, respectively. The 24-nt siRNA length dependence relies on the separation between the 5′-phosphorylated end of the guide RNA and dual cleavage sites formed by the paired ribonuclease III domains. These structural studies, complemented by functional data, provide insight into the dicing principle for Dicers in general.

Reader’s Corner Archive

Quote of January

“Imagination is more important than knowledge.”

Albert Einstein

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