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Highlights of Coronavirus Structural Studies

19 Feb 2021

Proton-Coupled Conformational Activation of SARS Coronavirus Main Proteases and Opportunity for Designing Small-Molecule Broad-Spectrum Targeted Covalent Inhibitors (JACS)

The SARS coronavirus 2 (SARS-CoV-2) main protease (Mpro) is an attractive broad-spectrum antiviral drug target. Despite the enormous progress in structureelucidation, the Mpro's structure-function relationship remains poorly understood. Recently, a peptidomimetic inhibitor has entered clinical trial; however, small-molecule orally available antiviral drugs have yet to be developed. Intrigued by a long-standing controversy regarding the existence of an inactive state, J. Shen et al. explored the proton-coupled dynamics of the Mpros of SARS-CoV-2 and the closely related SARS-CoV using a newly developed continuous constant pH molecular dynamics (MD) method and microsecond fixed-charge all-atom MD simulations. Their data supports a general base mechanism for Mpro's proteolytic function. The simulations revealed that protonation of His172 alters a conserved interaction network that upholds the oxyanion loop, leading to a partial collapse of the conserved S1 pocket, consistent with the first and controversial crystal structure of SARS-CoV Mpro determined at pH 6. Interestingly, a natural flavonoid binds SARS-CoV-2 Mpro in the close proximity to a conserved cysteine (Cys44), which is hyper-reactive according to the CpHMD titration. This finding offers an exciting new opportunity for small-molecule targeted covalent inhibitor design. Theirwork represents a first step toward the mechanistic understanding of the proton-coupled structure-dynamics-function relationship of CoV Mpros; the proposed strategy of designing small-molecule covalent inhibitors may help accelerate the development of orally available broad-spectrum antiviral drugs to stop the current pandemic and prevent future

19 Feb 2021

The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein (Nature Communications)

The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the similar to 30kb viral RNA genome to aid its packaging into the 80-90nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here D.W. Cleveland, K.D. Corbett  demonstrate that the N protein's central disordered domain drives phase separation with RNA, and that phosphorylation of an adjacent serine/arginine rich region modulates the physical properties of the resulting condensates. In cells, N forms condensates that recruit the stress granule protein G3BP1, highlighting a potential role for N in G3BP1 sequestration and stress granule inhibition. The SARS-CoV-2 membrane (M) protein independently induces N protein phase separation, and three-component mixtures of N+M+RNA form condensates with mutually exclusive compartments containing N+M or N+RNA, including annular structures in which the M protein coats the outside of an N+RNA condensate. These findings support a model in which phase separation of the SARS-CoV-2 N protein contributes both to suppression of the G3BP1-dependent host immune response and to packaging genomic RNA during virion assembly. The SARS-CoV-2 nucleocapsid (N) protein binds the viral RNA genome and contains two ordered domains flanked by three intrinsically-disordered regions. Here, the authors show that RNA binding induces liquid-liquid phase separation of N, which is driven by its central intrinsically-disordered region and is modulated by phosphorylation. The SARS-CoV-2 Membrane (M) protein also phase-separates with N, and three-component mixtures of N+M+RNA form mutually exclusive compartments containing N+M or N+RNA.

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Reader's Corner Archive

22 Jan 2020

Challenges and perspectives for structural biology of lncRNAs-the example of the Xist lncRNA A-repeats

Following the discovery of numerous long non-coding RNA (lncRNA) transcripts in the human genome, their important roles in biology and human disease are emerging. Recent progress in experimental methods has enabled the identification of structural features of lncRNAs. However, determining high-resolution structures is challenging as lncRNAs are expected to be dynamic and adopt multiple conformations, which may be modulated by interaction with protein binding partners. The X-inactive specific transcript (Xist) is necessary for X inactivation during dosage compensation in female placental mammals and one of the best-studied lncRNAs. Recent progress has provided new insights into the domain organization, molecular features, and RNA binding proteins that interact with distinct regions of Xist. The A-repeats located at the 5' end of the transcript are of particular interest as they are essential for mediating silencing of the inactive X chromosome. In this review, recent progress with elucidating structural features of the Xist lncRNA, focusing on the A-repeats is discussed. The experimental and computational approaches employed that have led to distinct structural models, likely reflecting the intrinsic dynamics of this RNA are overviewed. The presence of multiple dynamic conformations may also play an important role in the formation of the associated RNPs, thus influencing the molecular mechanism underlying the biological function of the Xist A-repeats. Alisha Jones and Michael Sattler in Journal of Molecular Cell Biology propose that integrative approaches that combine biochemical experiments and high-resolution structural biology in vitro with chemical probing and functional studies in vivo are required to unravel the molecular mechanisms of lncRNAs.

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 PLLx-b-PEGy 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/PLL1000–2000 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.

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