24 Sep 2020

SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation (Nat. Struct. Mol. Biol.)

The SARS-CoV-2 non-structural protein 1 (Nsp1), also referred to as the host shutoff factor, suppresses host innate immune functions. By combining cryo-electron microscopy and biochemistry, O. Mühlemann, N. Ban et.al.  show that SARS-CoV-2 Nsp1 binds to the human 40S subunit in ribosomal complexes, including the 43S pre-initiation complex and the non-translating 80S ribosome. The protein inserts its C-terminal domain into the mRNA channel, where it interferes with mRNA binding. They observe translation inhibition in the presence of Nsp1 in an in vitro translation system and in human cells. Based on the high-resolution structure of the 40S– Nsp1 complex, they identify residues of Nsp1 crucial for mediating translation inhibition. They further show that the full-length 5′ untranslated region of the genomic viral mRNA stimulates translation in vitro, suggesting that SARS-CoV-2 combines global inhibition of translation by Nsp1 with efficient translation of the viral mRNA to allow expression of viral genes.

28 Aug 2020

Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient (Nat. Struct Mol. Biol.)

The COVID-19 pandemic has had an unprecedented health and economic impact and there are currently no approved therapies. Stuart D., Huang, K.Y.A. et al. have isolated an antibody, EY6A, from an individual convalescing from COVID-19 and have shown that it neutralizes SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds the receptor binding domain (RBD) of the viral spike glycoprotein tightly (K(D)of 2 nM), and a 2.6-angstrom-resolution crystal structure of an RBD-EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues within this footprint are key to stabilizing the pre-fusion spike. Cryo-EM analyses of the pre-fusion spike incubated with EY6A Fab reveal a complex of the intact spike trimer with three Fabs bound and two further multimeric forms comprising the destabilized spike attached to Fab. EY6A binds what is probably a major neutralizing epitope, making it a candidate therapeutic for COVID-19. 

28 Aug 2020

Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike (Nature)

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic continues, with devasting consequences for human lives and the global economy. The discovery and development of virus-neutralizing monoclonal antibodies could be one approach to treat or prevent infection by this coronavirus. Here Huang, Y.X., Shapiro, L., and Ho, D.D. et.al. report the isolation of sixty-one SARS-CoV-2-neutralizing monoclonal antibodies from five patients infected with SARS-CoV-2 and admitted to hospital with severe coronavirus disease 2019 (COVID-19).Among these are nineteen antibodies that potently neutralized authentic SARS-CoV-2in vitro, nine of which exhibited very high potency, with 50% virus-inhibitory concentrations of 0.7 to 9 ng ml(-1). Epitope mapping showed that this collection of nineteen antibodies was about equally divided between those directed against the receptor-binding domain (RBD) and those directed against the N-terminal domain (NTD), indicating that both of these regions at the top of the viral spike are immunogenic. In addition, two other powerful neutralizing antibodies recognized quaternary epitopes that overlap with the domains at the top of the spike. Cryo-electron microscopy reconstructions of one antibody that targets the RBD, a second that targets the NTD, and a third that bridges two separate RBDs showed that the antibodies recognize the closed, 'all RBD-down' conformation of the spike. Several of these monoclonal antibodies are promising candidates for clinical development as potential therapeutic and/or prophylactic agents against SARS-CoV-2. 

13 Jul 2020

Structural basis of the activation of a metabotropic GABA receptor

Metabotropic gamma-aminobutyric acid receptors (GABA(B)) are involved in the modulation of synaptic responses in the central nervous system and have been implicated in neuropsychological conditions that range from addiction to psychosis. GABA(B)belongs to class C of the G-protein-coupled receptors, and its functional entity comprises an obligate heterodimer that is composed of the GB1 and GB2 subunits. Each subunit possesses an extracellular Venus flytrap domain, which is connected to a canonical seven-transmembrane domain. In the Nature paper Gati and Cherezov et.al. present four cryo-electron microscopy structures of the human full-length GB1-GB2 heterodimer: one structure of its inactive apo state, two intermediate agonist-bound forms and an active form in which the heterodimer is bound to an agonist and a positive allosteric modulator. The structures reveal substantial differences, which shed light on the complex motions that underlie the unique activation mechanism of GABA(B). Their results show that agonist binding leads to the closure of the Venus flytrap domain of GB1, triggering a series of transitions, first rearranging and bringing the two transmembrane domains into close contact along transmembrane helix 6 and ultimately inducing conformational rearrangements in the GB2 transmembrane domain via a lever-like mechanism to initiate downstream signalling. This active state is stabilized by a positive allosteric modulator binding at the transmembrane dimerization interface. 

13 Jul 2020

Structures of cell wall arabinosyltransferases with the anti-tuberculosis drug ethambutol

The arabinosyltransferases EmbA, EmbB, and EmbC are involved in Mycobacterium tuberculosis cell wall synthesis and are recognized as targets for the anti-tuberculosis drug ethambutol. In this study, published in Science, Wang, Li, Besra and Rao et.al. determined cryo–electron microscopy and x-ray crystal structures of mycobacterial EmbA-EmbB and EmbC-EmbC complexes in the presence of their glycosyl donor and acceptor substrates and with ethambutol. These structures show how the donor and acceptor substrates bind in the active site and how ethambutol inhibits arabinosyltransferases by binding to the same site as both substrates in EmbB and EmbC. Most drug-resistant mutations are located near the ethambutol binding site. Collectively, their work provides a structural basis for understanding the biochemical function and inhibition of arabinosyltransferases and the development of new anti-tuberculosis agents.

13 Jul 2020

Structural basis of DNA targeting by a transposon-encoded CRISPR-Cas system

Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements. Type I CRISPR–Cas systems typically target foreign DNA for degradation via joint action of the ribonucleoprotein complex Cascade and the helicase–nuclease Cas, but nuclease-deficient type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons. How CRISPR- and transposon-associated machineries collaborate during DNA targeting and insertion remains unknown. In the recent Nature publication Sternberg and Fernandez et al. describe structures of a TniQ–Cascade complex encoded by the Vibrio cholerae Tn6677 transposon using cryo-electron microscopy, revealing the mechanistic basis of this functional coupling. The cryo-electron microscopy maps enabled de novo modelling and refinement of the transposition protein TniQ, which binds to the Cascade complex as a dimer in a head-to-tail configuration, at the interface formed by Cas6 and Cas7 near the 3′ end of the CRISPR RNA (crRNA). The natural Cas8–Cas5 fusion protein binds the 5′ crRNA handle and contacts the TniQ dimer via a flexible insertion domain. A target DNA-bound structure reveals critical interactions necessary for protospacer-adjacent motif recognition and R-loop formation. This work lays the foundation for a structural understanding of how DNA targeting by TniQ–Cascade leads to downstream recruitment of additional transposase proteins, and will guide protein engineering efforts to leverage this system for programmable DNA insertions in genome-engineering applications.

7 Jul 2020

First Experiments in Structural Biology at the European X-ray Free-Electron Laser

Ultrabright pulses produced in X-ray free-electron lasers (XFELs) offer new possibilities for industry and research, particularly for biochemistry and pharmaceuticals. The unprecedented brilliance of these next-generation sources enables structure determination from sub-micron crystals as well as radiation-sensitive proteins. The European X-Ray Free-Electron Laser (EuXFEL), with its first light in 2017, ushered in a new era for ultrabright X-ray sources by providing an unparalleled megahertz-pulse repetition rate, with orders of magnitude more pulses per second than previous XFEL sources. This rapid pulse frequency has significant implications for structure determination; not only will data collection be faster (resulting in more structures per unit time), but experiments requiring large quantities of data, such as time-resolved structures, become feasible in a reasonable amount of experimental time. Early experiments at the SPB/SFX instrument of the EuXFEL demonstrate how such closely-spaced pulses can be successfully implemented in otherwise challenging experiments, such as time-resolved studies.

7 Jul 2020

In Situ Structure of an Intact Lipopolysaccharide-Bound Bacterial Surface Layer

Most bacterial and all archaeal cells are encapsulated by a paracrystalline, protective, and cell-shape-determining proteinaceous surface layer (S-layer). On Gram-negative bacteria, S-layers are anchored to cells via lipopolysaccharidevan. Kugelen et. al. report an electron cryo-microscopy structure of the Caulobacter crescentus S-layer bound to the O-antigen of lipopolysaccharide. Using native mass spectrometry and molecular dynamics simulations, they deduce the length of the O-antigen on cells and show how lipopolysaccharide binding and S-layer assembly is regulated by calcium. Finally, they present a near-atomic resolution in situ structure of the complete S-layer using cellular electron cryo-tomography, showing S-layer arrangement at the tip of the O-antigen. A complete atomic structure of the S-layer shows the power of cellular tomography for in situ structural biology and sheds light on a very abundant class of self-assembling molecules with important roles in prokaryotic physiology with marked potential for synthetic biology and surface-display applications.

7 Jul 2020

Toward Organism-scale Structural Biology: S-layer Reined in by Bacterial LPS

Technical developments are unifying molecular and cellular biology. A recent electron cryo-tomography study by von Kugelgen et al. highlights the bright future for such studies, seamlessly integrating near-atomic resolution protein structures, organism-scale architecture, native mass spectrometry, and molecular dynamic simulations to clarify how the Caulobacter crescentus S-layer assembles on the lipopolysaccharides (LPS) of the cell surface.

29 May 2020

Mapping Structural Dynamics of Proteins with Femtosecond Stimulated Raman Spectroscopy

The structure–function relationships of biomolecules have captured the interest and imagination of the scientific community and general public since the field of structural biology emerged to enable the molecular understanding of life processes. Proteins that play numerous functional roles in cellular processes have remained in the forefront of research, inspiring new characterization techniques. In this review in Annual Review of Physical Chemistry, Chong Fang and Longteng Tang present key theoretical concepts and recent experimental strategies using femtosecond stimulated Raman spectroscopy (FSRS) to map the structural dynamics of proteins, highlighting the flexible chromophores on ultrafast timescales. In particular, wavelength-tunable FSRS exploits dynamic resonance conditions to track transient-species-dependent vibrational motions, enabling rational design to alter functions. Various ways of capturing excited-state chromophore structural snapshots in the time and/or frequency domains are discussed. Continuous development of experimental methodologies, synergistic correlation with theoretical modeling, and the expansion to other nonequilibrium, photo-switchable, and controllable protein systems will greatly advance the chemical, physical, and biological sciences.

29 May 2020

The regulation and functions of DNA and RNA G-quadruplexes

DNA and RNA can adopt various secondary structures. Four-stranded G-quadruplex (G4) structures form through self-recognition of guanines into stacked tetrads, and considerable biophysical and structural evidence exists for G4 formation in vitro. Computational studies and sequencing methods have revealed the prevalence of G4 sequence motifs at gene regulatory regions in various genomes, including in humans. Experiments using chemical, molecular and cell biology methods have demonstrated that G4s exist in chromatin DNA and in RNA, and have linked G4 formation with key biological processes ranging from transcription and translation to genome instability and cancer. In the paper published in Nature Reviews Molecular Cell Biology, Balasubramanian, S. et al. first discuss the identification of G4s and evidence for their formation in cells using chemical biology, imaging and genomic technologies. They then discuss possible functions of DNA G4s and their interacting proteins, particularly in transcription, telomere biology and genome instability. Roles of RNA G4s in RNA biology, especially in translation, are also discussed. Furthermore, they consider the emerging relationships of G4s with chromatin and with RNA modifications. Finally, they discuss the connection between G4 formation and synthetic lethality in cancer cells, and recent progress towards considering G4s as therapeutic targets in human diseases.