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Highlights of Coronavirus Structural Studies

19 Feb

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

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 et.al.  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

8 Feb

Characterizing proteins in a native bacterial environment using solid-state NMR spectroscopy (Nature Protocols)

For a long time, solid-state nuclear magnetic resonance (ssNMR) has been employed to study complex biomolecular systems at the detailed chemical, structural, or dynamic level. Recent progress in high-resolution and high-sensitivity ssNMR, in combination with innovative sample preparation and labeling schemes, offers novel opportunities to study proteins in their native setting irrespective of the molecular tumbling rate. This protocol describes biochemical preparation schemes to obtain cellular samples of both soluble as well as insoluble or membrane-associated proteins in bacteria. To this end, the protocol is suitable for studying a protein of interest in both whole cells and in cell envelope or isolated membrane preparations. In the first stage of the procedure, an appropriate strain of Escherichia coli (DE3) is transformed with a plasmid of interest harboring the protein of interest under the control of an inducible T7 promoter. Next, the cells are adapted to grow in minimal (M9) medium. Before the growth enters stationary phase, protein expression is induced, and shortly thereafter, the native E. coli RNA polymerase is inhibited using rifampicin for targeted labeling of the protein of interest. The cells are harvested after expression and prepared for ssNMR rotor filling. In addition to conventional 13C/15N-detected ssNMR, we also outline how these preparations can be readily subjected to multidimensional ssNMR experiments using dynamic nuclear polarization (DNP) or proton (1H) detection schemes. M. Baldus et al. estimate that the entire preparative procedure until NMR experiments can be started takes 3–5 days.

1 Feb

Structure, self-assembly, and properties of a truncated reflectin variant (PNAS)

Naturally occurring and recombinant protein-based materials are frequently employed for the study of fundamental biological processes and are often leveraged for applications in areas as diverse as electronics, optics, bioengineering, medicine, and even fashion. Within this context, unique structural proteins known as reflectins have recently attracted substantial attention due to their key roles in the fascinating color-changing capabilities of cephalopods and their technological potential as biophotonic and bioelectronic materials. However, progress toward understanding reflectins has been hindered by their atypical aromatic and charged residue- enriched sequences, extreme sensitivities to subtle changes in environmental conditions, and well-known propensities for aggregation. Herein, A. Gorodetsky et.al. elucidate the structure of a reflectin variant at the molecular level, demonstrate a straightforward mechanical agitation-based methodology for controlling this variant’s hierarchical assembly, and establish a direct correlation between the protein’s structural characteristics and intrinsic optical properties. Altogether, their findings address multiple challenges associated with the development of reflectins as materials, furnish molecular-level insight into the mechanistic underpinnings of cephalopod skin cells’ color-changing functionalities, and may inform new research directions across biochemistry, cellular biology, bioengineering, and optics.

12 Jan

Small-molecule inhibitors of human mitochondrial DNA transcription (Nature)

Altered expression of mitochondrial DNA (mtDNA) occurs in ageing and a range of human pathologies (for example, inborn errors of metabolism, neurodegeneration and cancer). Here Claes M. Gustafsson, Nils-Göran Larsson et. al. describe first-in-class specific inhibitors of mitochondrial transcription (IMTs) that target the human mitochondrial RNA polymerase (POLRMT), which is essential for biogenesis of the oxidative phosphorylation (OXPHOS) system. The IMTs efficiently impair mtDNA transcription in a reconstituted recombinant system and cause a dose-dependent inhibition of mtDNA expression and OXPHOS in cell lines. To verify the cellular target, they performed exome sequencing of mutagenized cells and identified a cluster of amino acid substitutions in POLRMT that cause resistance to IMTs. They obtained a cryo-electron microscopy (cryo-EM) structure of POLRMT bound to an IMT, which further defined the allosteric binding site near the active centre cleft of POLRMT. The growth of cancer cells and the persistence of therapy-resistant cancer stem cells has previously been reported to depend on OXPHOS7-17, and they therefore investigated whether IMTs have anti-tumour effects. Four weeks of oral treatment with an IMT is well-tolerated in mice and does not cause OXPHOS dysfunction or toxicity in normal tissues, despite inducing a strong anti-tumour response in xenografts of human cancer cells. In summary, IMTs provide a potent and specific chemical biology tool to study the role of mtDNA expression in physiology and disease.

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