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.

27 Aug 2020

Half a century of amyloids: past, present and future (Chem. Rev.)

Amyloid diseases are global epidemics with profound health, social and economic implications and yet remain without a cure. This dire situation calls for research into the origin and pathological manifestations of amyloidosis to stimulate continued development of new therapeutics. In basic science and engineering, the cross-beta architecture has been a constant thread underlying the structural characteristics of pathological and functional amyloids, and realizing that amyloid structures can be both pathological and functional in nature has fueled innovations in artificial amyloids, whose use today ranges from water purification to 3D printing. At the conclusion of a half century since Eanes and Glenner's seminal study of amyloids in humans, this review commemorates the occasion by documenting the major milestones in amyloid research to date, from the perspectives of structural biology, biophysics, medicine, microbiology, engineering and nanotechnology. We also discuss new challenges and opportunities to drive this interdisciplinary field moving forward.

27 Aug 2020

NMR Methods for Structural Characterization of Protein-Protein Complexes (Front. Mol. Biosci.)

Protein-protein interactions and the complexes thus formed are critical elements in a wide variety of cellular events that require an atomic-level description to understand them in detail. Such complexes typically constitute challenging systems to characterize and drive the development of innovative biophysical methods. NMR spectroscopy techniques can be applied to extract atomic resolution information on the binding interfaces, intermolecular affinity, and binding-induced conformational changes in protein-protein complexes formed in solution, in the cell membrane, and in large macromolecular assemblies. Here Venditti V. et al. discuss experimental techniques for the characterization of protein-protein complexes in both solution NMR and solid-state NMR spectroscopy. The approaches include solvent paramagnetic relaxation enhancement and chemical shift perturbations (CSPs) for the identification of binding interfaces, and the application of intermolecular nuclear Overhauser effect spectroscopy and residual dipolar couplings to obtain structural constraints of protein-protein complexes in solution. Complementary methods in solid-state NMR are described, with emphasis on the versatility provided by heteronuclear dipolar recoupling to extract intermolecular constraints in differentially labeled protein complexes. The methods described are of particular relevance to the analysis of membrane proteins, such as those involved in signal transduction pathways, since they can potentially be characterized by both solution and solid-state NMR techniques, and thus outline key developments in this frontier of structural biology.

19 Aug 2020

Structure of human GABA(B) receptor in an inactive state (Nature)

The human GABA(B) receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction. A unique GPCR that is known to require heterodimerization for function, the GABA(B) receptor has two subunits, GABA(B1) and GABA(B2), that are structurally homologous but perform distinct and complementary functions. GABA(B1) recognizes orthosteric ligands, while GABA(B2) couples with G proteins. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail. Although the VFT heterodimer structure has been resolved, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here O.B. Clarke, J. Frank, Q.R. Fan et al. present a near full-length structure of the GABA(B) receptor at atomic resolution, captured in an inactive state by cryo-electron microscopy. Their structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. They also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity. The structure of the GABA(B) receptor in an inactive state reveals, amongst other features, a latch between the two subunits that locks the transmembrane domain interface, and the presence of large phospholipids that may modulate receptor function.

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.