9 Oct 2020

Feline coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication (Nat. Commun.)

The main protease, Mpro (or 3CLpro) in SARS-CoV-2 is a viable drug target because of its essential role in the cleavage of the virus polypeptide. Feline infectious peritonitis, a fatal coronavirus infection in cats, was successfully treated previously with a prodrug GC376, a dipeptide-based protease inhibitor. Here, M. J. Lemieux show the prodrug and its parent GC373, are effective inhibitors of the Mpro from both SARS-CoV and SARS-CoV-2 with IC50 values in the nanomolar range. Crystal structures of SARS-CoV-2 Mpro with these inhibitors have a covalent modification of the nucleophilic Cys145. NMR analysis reveals that inhibition proceeds via reversible formation of a hemithioacetal. GC373 and GC376 are potent inhibitors of SARS-CoV-2 replication in cell culture. They are strong drug candidates for the treatment of human coronavirus infections because they have already been successful in animals. The work here lays the framework for their use in human trials for the treatment of COVID-19.

9 Oct 2020

Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex (Cell)

SARS-CoV-2 is the causative agent of the 2019–2020 pandemic. The SARS-CoV-2 genome is replicated and transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here E.A.Campbell et. al. present cryo-electron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template product in complex with two molecules of the nsp13 helicase. The Nidovirales order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12 thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-trancribing holo-RdRp, constraining models for nsp13 function. They also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapy development.

24 Sep 2020

Growth, detection, quantification, and inactivation of SARS-CoV-2 (Virology)

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is the agent responsible for the coronavirus disease 2019 (COVID-19) global pandemic. SARS-CoV-2 is closely related to SARS-CoV, which caused the 2003 SARS outbreak. Although numerous reagents were developed to study SARS-CoV infections, few have been applicable to evaluating SARS-CoV-2 infection and immunity. Current limitations in studying SARS-CoV-2 include few validated assays with fully replication-competent wild-type virus. M.S. Diamond et. al.  have developed protocols to propagate, quantify, and work with infectious SARS-CoV-2. Here, we describe: (1) virus stock generation, (2) RT-qPCR quantification of SARS-CoV-2 RNA; (3) detection of SARS-CoV-2 antigen by flow cytometry, (4) quantification of infectious SARS-CoV-2 by focus-forming and plaque assays; and (5) validated protocols for virus inactivation. Collectively, these methods can be adapted to a variety of experimental designs, which should accelerate our understanding of SARS-CoV-2 biology and the development of effective countermeasures against COVID-19.

19 Aug 2020

Direct observation of dynamic protein interactions involving human microtubules using solid-state NMR spectroscopy (Nat. Commun.)

Microtubules are important components of the eukaryotic cytoskeleton. Their structural organization is regulated by nucleotide binding and many microtubule-associated proteins (MAPs). While cryo-EM and X-ray crystallography have provided detailed views of inter- actions between MAPs with the microtubule lattice, little is known about how MAPs and their intrinsically disordered regions interact with the dynamic microtubule surface. NMR carries the potential to directly probe such interactions but so far has been precluded by the low tubulin yield. M. Baldus et. al. present a protocol to produce [13C, 15N]-labeled, functional microtubules (MTs) from human cells for solid-state NMR studies. This approach allowed them to demonstrate that MAPs can differently modulate the fast time-scale dynamics of C-terminal tubulin tails, suggesting distinct interaction modes. Their results pave the way for in-depth NMR studies of protein dynamics involved in MT assembly and their interactions with other cellular components.

17 Aug 2020

Forced phage uncorking: viral DNA ejection triggered by a mechanically sensitive switch (Nanoscale)

The foremost event of bacteriophage infection is the ejection of genomic material into the host bacterium after virus binding to surface receptor sites. How ejection is triggered is yet unknown. Here M. S. Z. Kellermayer et.al. show, in single mature T7 phage particles, that tapping the capsid wall with an oscillating atomic-force-micro- scope cantilever triggers rapid DNA ejection via the tail complex. The triggering rate increases exponentially as a function of force, following transition-state theory, across an activation barrier of 23 kcal mol⁻¹ at 1.2 nm along the reaction coordinate. The conformation of the ejected DNA molecule revealed that it had been exposed to a propulsive force. This force, arising from intra-capsid pressure, assists in initiating the ejection process and the transfer of DNA across spatial dimensions beyond that of the virion. Chemical immobilization of the tail fibers also resulted in enhanced DNA ejection, suggesting that the triggering process might involve a conformational switch that can be mechanically activated either by external forces or via the tail-fiber complex.

11 Aug 2020

Structural insights into probe-dependent positive allosterism of the GLP-1 receptor (Nat. Chem. Biol.)

Drugs that promote the association of protein complexes are an emerging therapeutic strategy. K. Kobilka, K.W.Slopp et al. report discovery of a G protein-coupled receptor (GPCR) ligand that stabilizes an active state conformation by cooperatively binding both the receptor and orthosteric ligand, thereby acting as a ‘molecular glue’. LSN3160440 is a positive allosteric modulator of the GLP-1R optimized to increase the affinity and efficacy of GLP-1(9-36), a proteolytic product of GLP-1(7-36). The compound enhances insulin secretion in a glucose-, ligand- and GLP-1R-dependent manner. Cryo-electron microscopy determined the structure of the GLP-1R bound to LSN3160440 in complex with GLP-1 and heterotrimeric G_s. The modulator binds high in the helical bundle at an interface between TM1 and TM2, allowing access to the peptide ligand. Pharmacological characterization showed strong probe dependence of LSN3160440 for GLP-1(9-36) versus oxyntomodulin that is driven by a single residue. Their findings expand protein–protein modulation drug discovery to uncompetitive, active state stabilizers for peptide hormone receptors.

11 Aug 2020

Cryo-EM analysis of the post-fusion structure of the SARS-CoV spike glycoprotein (Nat. Commun.)

Global emergencies caused by the severe acute respiratory syndrome coronavirus (SARS- CoV), Middle-East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2 significantly endanger human health. The spike (S) glycoprotein is the key antigen and its conserved S2 subunit contributes to viral entry by mediating host-viral membrane fusion. However, structural information of the post-fusion S2 from these highly pathogenic human- infecting coronaviruses is still lacking. D. Cao, X. Zhang et. al. used single-particle cryo-electron microscopy to show that the post-fusion SARS-CoV S2 forms a further rotated HR1-HR2 six-helix bundle and a tightly bound linker region upstream of the HR2 motif. The structures of pre- and post- fusion SARS-CoV S glycoprotein dramatically differ, resembling that of the Mouse hepatitis virus (MHV) and other class I viral fusion proteins. This structure suggests potential targets for the development of vaccines and therapies against a wide range of SARS-like coronaviruses.

11 Aug 2020

CryoSIM: super-resolution 3D structured illumination cryogenic fluorescence microscopy for correlated ultrastructural imaging (Optica)

Rapid cryopreservation of biological specimens is the gold standard for visualizing cellular structures in their true structural context. However, current commercial cryo-fluorescence microscopes are limited to low resolutions. To fill this gap, I. M. Dobbie et. al. have developed cryoSIM, a microscope for 3D super-resolution fluorescence cryo-imaging for correlation with cryo-electron microscopy or cryo-soft X-ray tomography. They provide the full instructions for replicating the instrument mostly from off-the-shelf components and accessible, user-friendly, open-source Python control software. Therefore, cryoSIM democratizes the ability to detect molecules using super-resolution fluorescence imaging of cryopreserved specimens for correlation with their cellular ultrastructure.

29 Jul 2020

Solid-state NMR spectroscopy for characterization of RNA and RNP complexes

Ribonucleic acids are driving a multitude of biological processes where they act alone or in complex with proteins (ribonucleoproteins, RNP). To understand these processes both structural and mechanistic information about RNA is necessary. Due to their conformational plasticity RNA pose a challenge for mainstream structural biology methods. Solid- state NMR (ss-NMR) spectroscopy is an emerging technique that can be applied to biomolecular complexes of any size in close-to-native conditions. This review outlines recent methodological developments in ss-NMR for structural characterization of RNA and protein–RNA complexes and provides relevant examples.

29 Jul 2020

Mass spectrometry-based methods for structural biology on a proteome-wide scale

Mass spectrometry (MS) has long been used to study proteins mainly via sequence identification and quantitation of expression abundance. In recent years, MS has emerged as a tool for structural biology. Intact protein structural analysis has been enabled by the development of methods such as native MS, top-down proteomics, and ion mobility MS. Other MS-based structural methods include affinity purification MS, chemical cross-linking, and protein footprinting. These methods have enabled the study of protein–protein and protein–ligand interactions and regions of conformational change. The coupling of MS with liquid chromatography has permitted the analysis of complex samples. This bottom-up proteomics workflow enables the study of protein structure in the native cellular environment and provides structural information across the proteome. It has been demonstrated that the crowded environment of the cell affects protein binding interactions and affinities. Performing studies in this complex environment is essential for understanding the functional roles of proteins. MS-based structural methods permit analysis of samples such as cell lysates, intact cells, and tissue to provide a more physiological view of protein structure. This mini-review discusses the various MS-based methods that can be used for proteome-wide structural studies and highlights some of their application.

29 Jul 2020

The data universe of structural biology

The Protein Data Bank (PDB) has grown from a small data resource for crystallographers to a worldwide resource serving structural biology. The history of the growth of the PDB and the role that the community has played in developing standards and policies are described. This article also illustrates how other biophysics communities are collaborating with the worldwide PDB to create a network of interoperating data resources. This network will expand the capabilities of structural biology and enable the determination and archiving of increasingly complex structures.