30 Aug 2021

Structural and functional ramifications of antigenic drift in recent SARS-CoV-2 variants (Science)

Neutralizing antibodies (nAbs) elicited against the receptor binding site (RBS) of the spike protein of wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are generally less effective against recent variants of concern. RBS residues Glu(484), Lys(417), and Asn(501) are mutated in variants first described in South Africa (B.1.351) and Brazil (P.1). Wilson, I.A. et al.  analyzed their effects on angiotensin-converting enzyme 2 binding, as well as the effects of two of these mutations (K417N and E484K) on nAbs isolated from COVID-19 patients. Binding and neutralization of the two most frequently elicited antibody families (IGHV3-53/3-66 and IGHV1-2), which can both bind the RBS in alternative binding modes, are abrogated by K417N, E484K, or both. These effects can be structurally explained by their extensive interactions with RBS nAbs. However, nAbs to the more conserved, cross-neutralizing CR3022 and S309 sites were largely unaffected. The results have implications for next-generation vaccines and antibody therapies.

30 Aug 2021

Structural basis for enhanced infectivity and immune evasion of SARS-CoV-2 variants (Science)

Several fast-spreading variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have become the dominant circulating strains in the COVID-19 pandemic. Chen, B. et al. report here cryo-electron microscopy structures of the full-length spike (S) trimers of the B.1.1.7 (alfa) and B.1.351 (beta) variants, as well as their biochemical and antigenic properties. Amino acid substitutions in the B.1.1.7 protein increase both the accessibility of its receptor binding domain and the binding affinity for receptor angiotensin-converting enzyme 2 (ACE2). The enhanced receptor engagement may account for the increased transmissibility. The B.1.351 variant has evolved to reshape antigenic surfaces of the major neutralizing sites on the S protein, making it resistant to some potent neutralizing antibodies. These findings provide structural details on how SARS-CoV-2 has evolved to enhance viral fitness and immune evasion.

17 Aug 2021

Exploring the Druggability of Conserved RNA Regulatory Elements in the SARS-CoV-2 Genome (Angewandte Chemie Int. Ed.)

SARS-CoV-2 contains a positive single-stranded RNA genome of approximately 30 000 nucleotides. Within this genome, 15 RNA elements were identified as conserved between SARS-CoV and SARS-CoV-2. By nuclear magnetic resonance (NMR) spectroscopy, H. Schwalbe et al. previously determined that these elements fold independently, in line with data from in vivo and ex-vivo structural probing experiments. These elements contain non-base-paired regions that potentially harbor ligand-binding pockets. Here, they performed an NMR-based screening of a poised fragment library of 768 compounds for binding to these RNAs, employing three different ¹H-based 1D NMR binding assays. The screening identified common as well as RNA-element specific hits. The results allow selection of the most promising of the 15 RNA elements as putative drug targets. Based on the identified hits, they derive key functional units and groups in ligands for effective targeting of the RNA of SARS-CoV-2.

12 Jul 2021

Recent Developments in the Field of Intrinsically Disordered Proteins: Intrinsic Disorder-Based Emergence in Cellular Biology in Light of the Physiological and Pathological Liquid-Liquid Phase Transitions (Annual reviews of Biophysics)

This review deals with two important concepts-protein intrinsic disorder and proteinaceous membrane-less organelles (PMLOs). The past 20 years have seen an upsurge of scientific interest in these phenomena. However, neither are new discoveries made in this century, but instead are timely reincarnations of old ideas that were mostly ignored by the scientific community for a long time. Merging these concepts in the form of the intrinsic disorder-based biological liquid-liquid phase separation provides a basis for understanding the molecular mechanisms of PMLO biogenesis.

12 Jul 2021

Cryo-EM structure of the mature and infective Mayaro virus at 4.4 angstrom resolution reveals features of arthritogenic alphaviruses (Nature Communications)

Mayaro virus (MAYV) is an emerging arbovirus of the Americas that may cause a debilitating arthritogenic disease. The biology of MAYV is not fully understood and largely inferred from related arthritogenic alphaviruses. Here, we present the structure of MAYV at 4.4 angstrom resolution, obtained from a preparation of mature, infective virions. MAYV presents typical alphavirus features and organization. Interactions between viral proteins that lead to particle formation are described together with a hydrophobic pocket formed between E1 and E2 spike proteins and conformational epitopes specific of MAYV. We also describe MAYV glycosylation residues in E1 and E2 that may affect MXRA8 host receptor binding, and a molecular "handshake" between MAYV spikes formed by N262 glycosylation in adjacent E2 proteins. The structure of MAYV is suggestive of structural and functional complexity among alphaviruses, which may be targeted for specificity or antiviral activity. Mayaro virus (MAYV) is an emerging arbovirus in Central and South America that is transmitted by mosquitoes and causes arthritogenic disease. Here, the authors present the 4.4 angstrom resolution cryo-EM structure of MAYV and describe specific features of the virus, which could be exploited for the design of MAYV-specific diagnostics and therapeutics.

12 Jul 2021

Structural basis of omega-3 fatty acid transport across the blood-brain barrier (Nature)

Docosahexaenoic acid is an omega-3 fatty acid that is essential for neurological development and function, and it is supplied to the brain and eyes predominantly from dietary sources. This nutrient is transported across the blood-brain and blood-retina barriers in the form of lysophosphatidylcholine by major facilitator superfamily domain containing 2A (MFSD2A) in a Na+-dependent manner. Here we present the structure of MFSD2A determined using single-particle cryo-electron microscopy, which reveals twelve transmembrane helices that are separated into two pseudosymmetric domains. The transporter is in an inward-facing conformation and features a large amphipathic cavity that contains the Na+-binding site and a bound lysolipid substrate, which we confirmed using native mass spectrometry. Together with our functional analyses and molecular dynamics simulations, this structure reveals details of how MFSD2A interacts with substrates and how Na+-dependent conformational changes allow for the release of these substrates into the membrane through a lateral gate. Our work provides insights into the molecular mechanism by which this atypical major facility superfamily transporter mediates the uptake of lysolipids into the brain, and has the potential to aid in the delivery of neurotherapeutic agents.

18 May 2021

A viral genome packaging motor transitions between cyclic and helical symmetry to translocate dsDNA (Science Advances)

Molecular segregation and biopolymer manipulation require the action of molecular motors to do work by applying directional forces to macromolecules. The additional strand conserved E (ASCE) ring motors are an ancient family of molecular motors responsible for diverse biological polymer manipulation tasks. Viruses use ASCE segregation motors to package their genomes into their protein capsids and provide accessible experimental systems due to their relative simplicity. White, M.A., Jardine, P.J., Morais, M. C. et. al. show by cryo-EM–focused image reconstruction that ASCE ATPases in viral double-stranded DNA (dsDNA) packaging motors adopt helical symmetry complementary to their dsDNA substrates. Together with previous data, their results suggest that these motors cycle between helical and planar configurations, providing a possible mechanism for directional translocation of DNA. Similar changes in quaternary structure have been observed for proteasome and helicase motors, suggesting an ancient and common mechanism of force generation that has been adapted for specific tasks over the course of evolution.

18 May 2021

Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography (Nature Communications)

Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging of sub-volumes has been successfully used to derive atomic models of macromolecules in their biological environment. Eliminating biochemical isolation steps required by other techniques, this method opens up the cell to in-situ structural studies. However, the need to compensate for errors in targeting introduced during mechanical navigation of the specimen significantly slows down tomographic data collection thus limiting its practical value. Here, Borgnia, M. J., Bartesaghi, A. et. al. introduce protocols for tilt-series acquisition and processing that accelerate data collection speed by up to an order of magnitude and improve map resolution compared to existing approaches. They achieve this by using beam-image shift to multiply the number of areas imaged at each stage position, by integrating geometrical constraints during imaging to achieve high precision targeting, and by performing per-tilt astigmatic CTF estimation and data-driven exposure weighting to improve final map resolution. They validated our beam image-shift electron cryo-tomography (BISECT) approach by determining the structure of a low molecular weight target (~300 kDa) at 3.6 Å resolution where density for individual side chains is clearly resolved.

18 May 2021

VESPER: global and local cryo-EM map alignment using local density vectors (Nature Communications)

An increasing number of density maps of biological macromolecules have been determined by cryo-electron microscopy (cryo-EM) and stored in the public database, EMDB. To interpret the structural information contained in EM density maps, alignment of maps is an essential step for structure modeling, comparison of maps, and for database search. Here, Kihara, D. et al. developed VESPER, which captures the similarity of underlying molecular structures embedded in density maps by taking local gradient directions into consideration.Compared to existing methods, VESPER achieved substantially more accurate global and local alignment of maps as well as database retrieval.

4 May 2021

Mechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism (Nature Communications)

Bacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, R. C. J. Dobson et. al. demonstrate that three NanR dimers bind a (GGTATA)(3)-repeat operator cooperatively and with high affinity. Single-particle cryo-electron microscopy structures reveal the DNA-binding domain is reorganized to engage DNA, while three dimers assemble in close proximity across the (GGTATA)(3)-repeat operator. Such an interaction allows cooperative protein-protein interactions between NanR dimers via their N-terminal extensions. The effector, N-acetylneuraminate, binds NanR and attenuates the NanR-DNA interaction. The crystal structure of NanR in complex with N-acetylneuraminate reveals a domain rearrangement upon N-acetylneuraminate binding to lock NanR in a conformation that weakens DNA binding. Our data provide a molecular basis for the regulation of bacterial sialic acid metabolism. The GntR superfamily is one of the largest families of transcription factors in prokaryotes. Here the authors combine biophysical analysis and structural biology to dissect the mechanism by which NanR - a GntR-family regulator - binds to its promoter to repress the transcription of genes necessary for sialic acid metabolism.

4 May 2021

Single-molecule FRET imaging of GPCR dimers in living cells (Nature Methods)

Class C G protein-coupled receptors (GPCRs) are known to form stable homodimers or heterodimers critical for function, but the oligomeric status of class A and B receptors, which constitute >90% of all GPCRs, remains hotly debated. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful approach with the potential to reveal valuable insights into GPCR organization but has rarely been used in living cells to study protein systems. Here, J. A. Javitch et. al. report generally applicable methods for using smFRET to detect and track transmembrane proteins diffusing within the plasma membrane of mammalian cells. They leverage this in-cell smFRET approach to show agonist-induced structural dynamics within individual metabotropic glutamate receptor dimers. They apply these methods to representative class A, B and C receptors, finding evidence for receptor monomers, density-dependent dimers and constitutive dimers, respectively.