24 Sep 2020

SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation (Nat. Struct. Mol. Biol.)

The SARS-CoV-2 non-structural protein 1 (Nsp1), also referred to as the host shutoff factor, suppresses host innate immune functions. By combining cryo-electron microscopy and biochemistry, O. Mühlemann, N. Ban et.al.  show that SARS-CoV-2 Nsp1 binds to the human 40S subunit in ribosomal complexes, including the 43S pre-initiation complex and the non-translating 80S ribosome. The protein inserts its C-terminal domain into the mRNA channel, where it interferes with mRNA binding. They observe translation inhibition in the presence of Nsp1 in an in vitro translation system and in human cells. Based on the high-resolution structure of the 40S– Nsp1 complex, they identify residues of Nsp1 crucial for mediating translation inhibition. They further show that the full-length 5′ untranslated region of the genomic viral mRNA stimulates translation in vitro, suggesting that SARS-CoV-2 combines global inhibition of translation by Nsp1 with efficient translation of the viral mRNA to allow expression of viral genes.

28 Aug 2020

Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient (Nat. Struct Mol. Biol.)

The COVID-19 pandemic has had an unprecedented health and economic impact and there are currently no approved therapies. Stuart D., Huang, K.Y.A. et al. have isolated an antibody, EY6A, from an individual convalescing from COVID-19 and have shown that it neutralizes SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds the receptor binding domain (RBD) of the viral spike glycoprotein tightly (K(D)of 2 nM), and a 2.6-angstrom-resolution crystal structure of an RBD-EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues within this footprint are key to stabilizing the pre-fusion spike. Cryo-EM analyses of the pre-fusion spike incubated with EY6A Fab reveal a complex of the intact spike trimer with three Fabs bound and two further multimeric forms comprising the destabilized spike attached to Fab. EY6A binds what is probably a major neutralizing epitope, making it a candidate therapeutic for COVID-19. 

28 Aug 2020

Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike (Nature)

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic continues, with devasting consequences for human lives and the global economy. The discovery and development of virus-neutralizing monoclonal antibodies could be one approach to treat or prevent infection by this coronavirus. Here Huang, Y.X., Shapiro, L., and Ho, D.D. et.al. report the isolation of sixty-one SARS-CoV-2-neutralizing monoclonal antibodies from five patients infected with SARS-CoV-2 and admitted to hospital with severe coronavirus disease 2019 (COVID-19).Among these are nineteen antibodies that potently neutralized authentic SARS-CoV-2in vitro, nine of which exhibited very high potency, with 50% virus-inhibitory concentrations of 0.7 to 9 ng ml(-1). Epitope mapping showed that this collection of nineteen antibodies was about equally divided between those directed against the receptor-binding domain (RBD) and those directed against the N-terminal domain (NTD), indicating that both of these regions at the top of the viral spike are immunogenic. In addition, two other powerful neutralizing antibodies recognized quaternary epitopes that overlap with the domains at the top of the spike. Cryo-electron microscopy reconstructions of one antibody that targets the RBD, a second that targets the NTD, and a third that bridges two separate RBDs showed that the antibodies recognize the closed, 'all RBD-down' conformation of the spike. Several of these monoclonal antibodies are promising candidates for clinical development as potential therapeutic and/or prophylactic agents against SARS-CoV-2. 

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.

13 Jul 2020

Structural basis of the activation of a metabotropic GABA receptor

Metabotropic gamma-aminobutyric acid receptors (GABA(B)) are involved in the modulation of synaptic responses in the central nervous system and have been implicated in neuropsychological conditions that range from addiction to psychosis. GABA(B)belongs to class C of the G-protein-coupled receptors, and its functional entity comprises an obligate heterodimer that is composed of the GB1 and GB2 subunits. Each subunit possesses an extracellular Venus flytrap domain, which is connected to a canonical seven-transmembrane domain. In the Nature paper Gati and Cherezov et.al. present four cryo-electron microscopy structures of the human full-length GB1-GB2 heterodimer: one structure of its inactive apo state, two intermediate agonist-bound forms and an active form in which the heterodimer is bound to an agonist and a positive allosteric modulator. The structures reveal substantial differences, which shed light on the complex motions that underlie the unique activation mechanism of GABA(B). Their results show that agonist binding leads to the closure of the Venus flytrap domain of GB1, triggering a series of transitions, first rearranging and bringing the two transmembrane domains into close contact along transmembrane helix 6 and ultimately inducing conformational rearrangements in the GB2 transmembrane domain via a lever-like mechanism to initiate downstream signalling. This active state is stabilized by a positive allosteric modulator binding at the transmembrane dimerization interface. 

13 Jul 2020

Structures of cell wall arabinosyltransferases with the anti-tuberculosis drug ethambutol

The arabinosyltransferases EmbA, EmbB, and EmbC are involved in Mycobacterium tuberculosis cell wall synthesis and are recognized as targets for the anti-tuberculosis drug ethambutol. In this study, published in Science, Wang, Li, Besra and Rao et.al. determined cryo–electron microscopy and x-ray crystal structures of mycobacterial EmbA-EmbB and EmbC-EmbC complexes in the presence of their glycosyl donor and acceptor substrates and with ethambutol. These structures show how the donor and acceptor substrates bind in the active site and how ethambutol inhibits arabinosyltransferases by binding to the same site as both substrates in EmbB and EmbC. Most drug-resistant mutations are located near the ethambutol binding site. Collectively, their work provides a structural basis for understanding the biochemical function and inhibition of arabinosyltransferases and the development of new anti-tuberculosis agents.