24 Aug 2020

Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19 (Cell)

SARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. Here M. Buggert et al. systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T cell responses in unexposed individuals, exposed family members, and individuals with acute or convalescent COVID-19. Acute phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative exposed family members and convalescent individuals with a history of asymptomatic and mild COVID-19. Their collective dataset shows that SARS-CoV-2 elicits robust, broad and highly functional memory T cell responses, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19.

19 Aug 2020

Vaccine candidate protects monkeys from SARS-CoV-2 infection (Nature)

An experimental coronavirus vaccine Ad26 seems to have completely prevented infection in most monkeys that received the jab. Hanneke Schuitemaker at Janssen Vaccines and Prevention in Leiden, the Netherlands, Dan Barouch at Beth Israel Deaconess Medical Center in Boston, Massachusetts, and their colleagues gave 52 rhesus macaques (Macaca mulatta) a single dose of one of 7 vaccines (N. B. Mercado et al. Nature 2020). Each vaccine comprised a weakened respiratory virus coding for one of seven forms of SARS-CoV-2’s spike protein. After vaccination, nearly all the monkeys made neutralizing antibodies — powerful immune molecules that can block infection — and T cells that trigger other immune responses. When monkeys were exposed to SARS-CoV-2, the most potent of the vaccines Ad26 prevented lung infection in six out of six animals that received it using intratracheal route, and nasal infection in five out of six.Across all the vaccinated monkeys, levels of neutralizing antibodies were associated with protection from SARS-CoV-2 infection, but levels of T cells were not. The optimal Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is currently being evaluated in clinical trials.

18 Aug 2020

Human neutralizing antibodies elicited by SARS-CoV-2 infection (Nature)

The emerging coronavirus SARS-CoV-2 pandemic presents a global health emergency in urgent need of interventions. SARS-CoV-2 entry into the target cells depends on binding between the receptor-binding domain (RBD) of the viral Spike protein and the ACE2 cell receptor. Here, Wang, Z. Zhang, L. Zhang et. al.  report the isolation and characterization of 206 RBD-specific monoclonal antibodies derived from single B cells of eight SARS-CoV-2 infected individuals. They identified antibodies with potent anti-SARS-CoV-2 neutralization activity that correlates with their competitive capacity with ACE2 for RBD binding. Surprisingly, neither the anti-SARS-CoV-2 antibodies nor the infected plasma cross-reacted with SARS-CoV or MERS-CoV RBDs, although substantial plasma cross-reactivity to their trimeric Spike proteins was found. Crystal structure analysis of RBD-bound antibody revealed steric hindrance that inhibits viral engagement with ACE2 and thereby blocks viral entry. These findings suggest that anti-RBD antibodies are viral species-specific inhibitors. The antibodies identified here may be candidates for the development of SARS-CoV-2 clinical interventions.

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.

29 May 2020

The architecture of the Gram-positive bacterial cell wall

The primary structural component of the bacterial cell wall is peptidoglycan, which is essential for viability and the synthesis of which is the target for crucial antibiotics. Peptidoglycan is a single macromolecule made of glycan chains crosslinked by peptide side branches that surrounds the cell, acting as a constraint to internal turgor. In Gram-positive bacteria, peptidoglycan is tens of nanometers thick, generally portrayed as a homogeneous structure that provides mechanical strength.  S. J. Foster &  J. K. Hobbs et.al. applied atomic force microscopy to interrogate the morphologically distinct Staphylococcus aureus and Bacillus subtilis species, using live cells and purified peptidoglycan. The paper published in Nature shows that the mature surface of live cells is characterized by a landscape of large (up to 60 nm in diameter), deep (up to 23 nm) pores constituting a disordered gel of peptidoglycan. The inner peptidoglycan surface, consisting of more nascent material, is much denser, with glycan strand spacing typically less than 7 nm. The inner surface architecture is location dependent; the cylinder of B. subtilis has dense circumferential orientation, while in S. aureus and division septa for both species, peptidoglycan is dense but randomly oriented. Revealing the molecular architecture of the cell envelope frames our understanding of its mechanical properties and role as the environmental interface, providing information complementary to traditional structural biology approaches.

15 May 2020

Emerging solution NMR methods to illuminate the structural and dynamic properties of proteins

The first recognition of protein breathing was more than 50 years ago. Today, we are able to detect the multitude of interaction modes, structural polymorphisms, and binding-induced changes in protein structure that direct function. Solution-state NMR spectroscopy has proved to be a powerful technique, not only to obtain high-resolution structures of proteins, but also to provide unique insights into the functional dynamics of proteins. Hari Arthanari, Gerhard Wager et. al. in the Current Opinion in Structural Biology review summarize recent technical landmarks in solution NMR that have enabled characterization of key biological macromolecular systems. These methods have been fundamental to atomic resolution structure determination and quantitative analysis of dynamics over a wide range of time scales by NMR. The ability of NMR to detect lowly populated protein conformations and transiently formed complexes plays a critical role in its ability to elucidate functionally important structural features of proteins and their dynamics.