29 Jul

SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects

SARS-CoV-2 is thought to have emerged from bats, possibly via a secondary host. Here, we investigate the relationship of spike (S) glycoprotein from SARS-CoV-2 with the S protein of a closely related bat virus, RaTG13. Antoni Wrobel, Donald Benton, Steven Gamblin et al.  determined cryo-EM structures for RaTG13 S and for both furin-cleaved and uncleaved SARS-CoV-2 S; they compared these with recently reported structures for uncleaved SARS-CoV-2 S. They also biochemically characterized their relative stabilities and affinities for the SARS-CoV-2 receptor ACE2. Although the overall structures of human and bat virus S proteins are similar, there are key differences in their properties, including a more stable precleavage form of human S and about 1,000-fold tighter binding of SARS-CoV-2 to human receptor. These observations suggest that cleavage at the furin-cleavage site decreases the overall stability of SARS-CoV-2 S and facilitates the adoption of the open conformation that is required for S to bind to the ACE2 receptor. 

29 Jul

Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2

Two nanobodies that bind SARS-CoV-2 spike RBD are shown to block interaction with receptor ACE2 and thus neutralize the virus, and have an additive effect with antibody CR3022. The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, Raymond Owens, James Naismith et al. have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (K(D)of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.

17 Jul

Stabilizing the Closed SARS-CoV-2 Spike Trimer

The trimeric spike (S) protein of SARS-CoV-2 is the primary focus of most vaccine design and development efforts. Due to intrinsic instability typical of class I fusion proteins, S tends to prematurely refold to the post-fusion conformation, compromising immunogenic properties and prefusion trimer yields. To support ongoing vaccine development efforts, P.M. Langedijk et al. report the structure-based design of soluble S trimers, with increased yields and stabilities, based on introduction of single point mutations and disulfide-bridges. They identify two regions in the S-protein critical for the protein’s stability: the heptad repeat region 1 of the S2 subunit and subunit domain 1 at the interface with S2. We combined a minimal selection of mostly inter-protomeric mutations to create a stable S-closed variant with a 6.4-fold higher expression than the parental construct while no longer containing a heterologous trimerization domain. The cryo-EM structure reveals a correctly folded, predominantly closed pre-fusion conformation. Highly stable and well producing S protein and the increased understanding of S protein structure will support vaccine development and serological diagnostics.

3 Dec 2019

PEGylated surfaces for the study of DNA–protein interactions by atomic force microscopy

DNA–protein interactions are vital to cellular function, with key roles in the regulation of gene expression and genome maintenance. Atomic force microscopy (AFM) offers the ability to visualize DNA–protein interactions at nanometer resolution in near-physiological buffers, but it requires that the DNA be adhered to the surface of a solid substrate. This presents a problem when working in biologically relevant protein concentrations, where proteins may be present in large excess in solution; much of the biophysically relevant information can therefore be occluded by non-specific protein binding to the underlying substrate. Here we explore the use of PLL_x-b-PEG_y block copolymers to achieve selective adsorption of DNA on a mica surface for AFM studies. Through varying both the number of lysine and ethylene glycol residues in the block copolymers, Bart Hoogenboom, Alice Pyne et al. show selective adsorption of DNA on mica that is functionalized with a PLL₁₀-b-PEG₁₁₃/PLL_1000–2000 mixture as viewed by AFM imaging in a solution containing high concentrations of streptavidin. They demonstrate – through the use of biotinylated DNA and streptavidin – that this selective adsorption extends to DNA–protein complexes and that DNA-bound streptavidin can be unambiguously distinguished in spite of an excess of unbound streptavidin in solution. Finally, they apply this to the nuclear enzyme PARP1, resolving the binding of individual PARP1 molecules to DNA by in-liquid AFM.

28 Nov 2019

More than Proton Detection—New Avenues for NMR Spectroscopy of RNA

This minireview written by Harald Schwalbe, Boris Fürtig and coworkers reports on the development of NMR methods that utilize detection on low-g nuclei (heteronuclei like ¹³C or ¹⁵N with lower gyromagnetic ratio than ¹H) to obtain unique structural and dynamic information for large RNA molecules in solution. Experiments involve through-bond correlations of nucleobases and the phosphodiester backbone of RNA for chemical shift assignment and make information on hydrogen bonding uniquely accessible. Previously unobservable NMR resonances of amino groups in RNA nucleobases are now detected in experiments involving conformational exchange-resistant double-quantum ¹H coherences, detected by ¹³C NMR spectroscopy. Furthermore, ¹³C and ¹⁵N chemical shifts provide valuable information on conformations. All the covered aspects point to the advantages of low-g nuclei detection experiments in RNA.

28 Nov 2019

Toward high-resolution in situ structural biology with cryo-electron tomography and subtomogram averaging

Cryo-electron tomography (cryo-ET) provides unprecedented insights into the molecular constituents of biological environments. In combination with an image processing method called subtomogram averaging (STA), detailed 3D structures of biological molecules can be obtained in large, irregular macromolecular assemblies or in situ, without the need for purification. The contextual meta-information these methods also provide, such as a protein’s location within its native environment, can then be combined with functional data. This allows the derivation of a detailed view on the physiological or pathological roles of proteins from the molecular to cellular level. Despite their tremendous potential in in situ structural biology, cryo-ET and STA have been restricted by methodological limitations, such as the low obtainable resolution. Exciting progress now allows one to reach unprecedented resolutions in situ, ranging in optimal cases beyond the nanometer barrier. Here, Florian Schur reviews current frontiers and future challenges in routinely determining high-resolution structures in in situ environments using cryo-ET and STA.

31 Oct 2019

Chemical cross-linking with mass spectrometry: a tool for systems structural biology

Biological processes supporting life are orchestrated by a highly dynamic array of protein structures and interactions comprising the interactome. Defining the interactome, visualizing how structures and interactions change and function to support life is essential to improved understanding of fundamental molecular processes, but represents a challenge unmet by any single analytical technique. Chemical cross-linking with mass spectrometry provides identification of proximal amino acid residues within proteins and protein complexes, yielding low resolution structural information. This approach has predominantly been employed to provide structural insight on isolated protein complexes, and has been particularly useful for molecules that are recalcitrant to conventional structural biology studies. In this review, Juan D. Chavez and James E. Bruce discuss recent developments in cross-linking and mass spectrometry technologies that are providing large-scale or systems-level interactome data with successful applications to isolated organelles, cell lysates, virus particles, intact bacterial and mammalian cultured cells and tissue samples.

30 Oct 2019

In‐Cell EPR: Progress towards Structural Studies Inside Cells

Exploring the structure and dynamics of biomolecules in the context of their intracellular environment has become the ultimate challenge for structural biology. As the cellular environment is barely reproducible in vitro, investigation of biomolecules directly inside cells has attracted a growing interest. Among magnetic resonance approaches, site‐directed spin labeling (SDSL) coupled to electron paramagnetic resonance (EPR) spectroscopy provides competitive and advantageous features to capture protein structure and dynamics inside cells. To date, several in‐cell EPR approaches have been successfully applied to both bacterial and eukaryotic cells. In this minireview, the major advances of in‐cell EPR spectroscopy are summarized, as well as the challenges this approach still poses.

30 Sep 2019

A standardized citation metrics author database annotated for scientific field

Citation metrics are widely used and misused. John P. A. Ioannidis et. al. created a publicly available data-base of 100,000 top scientists that provides standardized information on citations, h-index, co-authorship-adjusted hm-index, citations to papers in different authorship positions, and a composite indicator. Separate data are shown for career-long and single-year impact. Metrics with and without self-citations and ratio of citations to citing papers are given. Scientists are classified into 22 scientific fields and 176 subfields. Field- and subfield-specific percentiles are also provided for all scientists who have published at least five papers. Career-long data are updated to end of 2017 and to end of 2018 for comparison.

30 Sep 2019

How structure informs and transforms chemogenetics

Chemogenetic technologies such as Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are widely used to remotely control neuronal and non-neuronal signaling. DREADDs exist for most of the canonical G protein-coupled receptor signaling pathways, and provide a synthetic biology platform useful for elucidating the role of neuronal signaling for brain function. Here, Bryan L. Roth presents a focused review that shows how recent insights obtained from GPCR structural studies inform our understanding of these chemogenetic tools from a structural perspective.

12 Sep 2019

Emerging structural insights into glycosyltransferase-mediated synthesis of glycans

Glycans linked to proteins and lipids play key roles in biology; thus, accurate replication of cellular glycans is crucial for maintaining function following cell division.  Several recent crystal structures of glycosyltransferases with bound acceptor substrates reveal that these enzymes have common core structures that function as scaffolds upon which variable loops are inserted to confer substrate specificity and correctly orient the nucleophilic hydroxyl group. K. W. Moremen and R. S. Haltiwanger in Nature Chemical Biology review argue that the varied approaches for acceptor binding site assembly suggest that an ongoing evolution of these loop regions provides templates for assembly of the diverse glycan structures observed in biology.