Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by virus binding to the ACE2 cell-surface receptors
, followed by fusion of the virus and cell membranes to ...release the virus genome into the cell. Both receptor binding and membrane fusion activities are mediated by the virus spike glycoprotein
. As with other class-I membrane-fusion proteins, the spike protein is post-translationally cleaved, in this case by furin, into the S1 and S2 components that remain associated after cleavage
. Fusion activation after receptor binding is proposed to involve the exposure of a second proteolytic site (S2'), cleavage of which is required for the release of the fusion peptide
. Here we analyse the binding of ACE2 to the furin-cleaved form of the SARS-CoV-2 spike protein using cryo-electron microscopy. We classify ten different molecular species, including the unbound, closed spike trimer, the fully open ACE2-bound trimer and dissociated monomeric S1 bound to ACE2. The ten structures describe ACE2-binding events that destabilize the spike trimer, progressively opening up, and out, the individual S1 components. The opening process reduces S1 contacts and unshields the trimeric S2 core, priming the protein for fusion activation and dissociation of ACE2-bound S1 monomers. The structures also reveal refolding of an S1 subdomain after ACE2 binding that disrupts interactions with S2, which involves Asp614
and leads to the destabilization of the structure of S2 proximal to the secondary (S2') cleavage site.
The 1918 influenza pandemic resulted in about 20 million deaths. This enormous impact, coupled with renewed interest in emerging infections, makes characterization of the virus involved a priority. ...Receptor binding, the initial event in virus infection, is a major determinant of virus transmissibility that, for influenza viruses, is mediated by the hemagglutinin (HA) membrane glycoprotein. We have determined the crystal structures of the HA from the 1918 virus and two closely related HAs in complex with receptor analogs. They explain how the 1918 HA, while retaining receptor binding site amino acids characteristic of an avian precursor HA, is able to bind human receptors and how, as a consequence, the virus was able to spread in the human population.
SARS-CoV-2 is thought to have emerged from bats, possibly via a secondary host. Here, we investigate the relationship of spike (S) glycoprotein from SARS-CoV-2 with the S protein of a closely related ...bat virus, RaTG13. We determined cryo-EM structures for RaTG13 S and for both furin-cleaved and uncleaved SARS-CoV-2 S; we compared these with recently reported structures for uncleaved SARS-CoV-2 S. We also biochemically characterized their relative stabilities and affinities for the SARS-CoV-2 receptor ACE2. Although the overall structures of human and bat virus S proteins are similar, there are key differences in their properties, including a more stable precleavage form of human S and about 1,000-fold tighter binding of SARS-CoV-2 to human receptor. These observations suggest that cleavage at the furin-cleavage site decreases the overall stability of SARS-CoV-2 S and facilitates the adoption of the open conformation that is required for S to bind to the ACE2 receptor.
Influenza virus remains a threat because of its ability to evade vaccine-induced immune responses due to antigenic drift. Here, we describe the isolation, evolution, and structure of a broad-spectrum ...human monoclonal antibody (mAb), MEDI8852, effectively reacting with all influenza A hemagglutinin (HA) subtypes. MEDI8852 uses the heavy-chain VH6-1 gene and has higher potency and breadth when compared to other anti-stem antibodies. MEDI8852 is effective in mice and ferrets with a therapeutic window superior to that of oseltamivir. Crystallographic analysis of Fab alone or in complex with H5 or H7 HA proteins reveals that MEDI8852 binds through a coordinated movement of CDRs to a highly conserved epitope encompassing a hydrophobic groove in the fusion domain and a large portion of the fusion peptide, distinguishing it from other structurally characterized cross-reactive antibodies. The unprecedented breadth and potency of neutralization by MEDI8852 support its development as immunotherapy for influenza virus-infected humans.
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•Binding to all influenza A subtypes neutralizing seasonal and pandemic strains•Utilizes a rare VH (VH6-1) and carries a low level of somatic mutations•Highly conserved epitope encompassing fusion peptide and hydrophobic groove•Superior therapeutic window compared to oseltamivir in animals
Identification of a human monoclonal antibody that reacts effectively with all influenza A hemagglutinin subtypes paves the way for developing immunotherapy for people infected with the flu virus.
Covalent DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with essential chromatin transactions, such as replication and transcription. Little was known about DPC-specific repair ...mechanisms until the recent identification of a DPC-processing protease in yeast. The existence of a DPC protease in higher eukaryotes is inferred from data in Xenopus laevis egg extracts, but its identity remains elusive. Here we identify the metalloprotease SPRTN as the DPC protease acting in metazoans. Loss of SPRTN results in failure to repair DPCs and hypersensitivity to DPC-inducing agents. SPRTN accomplishes DPC processing through a unique DNA-induced protease activity, which is controlled by several sophisticated regulatory mechanisms. Cellular, biochemical, and structural studies define a DNA switch triggering its protease activity, a ubiquitin switch controlling SPRTN chromatin accessibility, and regulatory autocatalytic cleavage. Our data also provide a molecular explanation on how SPRTN deficiency causes the premature aging and cancer predisposition disorder Ruijs-Aalfs syndrome.
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•The SPRTN metalloprotease repairs DNA-protein crosslinks•A DNA switch controls SPRTN’s protease activity•A ubiquitin switch controls chromatin access of SPRTN•Structural insights reveal unique features of the SPRTN/Wss1 protease family
Stingele et al. discover the SPRTN metalloprotease to be crucial for DNA-protein crosslink repair in higher eukaryotes. In addition, several regulatory principles constraining SPRTN’s potentially toxic activity are described: a ubiquitin switch controlling chromatin access, a DNA switch triggering protease activity, and a negative feedback loop based on autocatalytic cleavage.
Hemagglutinin (HA) is the receptor-binding and membrane fusion glycoprotein
of influenza virus and the target for infectivity-neutralizing antibodies. The
structures of three conformations of the ...ectodomain of the 1968 Hong Kong
influenza virus HA have been determined by X-ray crystallography: the
single-chain precursor, HA0; the metastable neutral-pH conformation found on
virus, and the fusion pH-induced conformation. These structures provide a
framework for designing and interpreting the results of experiments on the
activity of HA in receptor binding, the generation of emerging and reemerging
epidemics, and membrane fusion during viral entry. Structures of HA in complex
with sialic acid receptor analogs, together with binding experiments, provide
details of these low-affinity interactions in terms of the sialic acid
substituents recognized and the HA residues involved in recognition.
Neutralizing antibody-binding sites surround the receptor-binding pocket on the
membrane-distal surface of HA, and the structures of the complexes between
neutralizing monoclonal Fabs and HA indicate possible neutralization
mechanisms. Cleavage of the biosynthetic precursor HA0 at a prominent loop in
its structure primes HA for subsequent activation of membrane fusion at
endosomal pH (
Figure 1
). Priming involves
insertion of the fusion peptide into a charged pocket in the precursor;
activation requires its extrusion towards the fusion target membrane, as the N
terminus of a newly formed trimeric coiled coil, and repositioning of the
C-terminal membrane anchor near the fusion peptide at the same end of a
rod-shaped molecule. Comparison of this new HA conformation, which has been
formed for membrane fusion, with the structures determined for other virus
fusion glycoproteins suggests that these molecules are all in the
fusion-activated conformation and that the juxtaposition of the membrane anchor
and fusion peptide, a recurring feature, is involved in the fusion mechanism.
Extension of these comparisons to the soluble N-ethyl-maleimide-sensitive
factor attachment protein receptor (SNARE) protein complex of vesicle fusion
allows a similar conclusion.
The majority of currently circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses have mutant spike glycoproteins that contain the D614G substitution. Several studies have ...suggested that spikes with this substitution are associated with higher virus infectivity. We use cryo-electron microscopy to compare G614 and D614 spikes and show that the G614 mutant spike adopts a range of more open conformations that may facilitate binding to the SARS-CoV-2 receptor, ACE2, and the subsequent structural rearrangements required for viral membrane fusion.
The efficient and timely resolution of DNA recombination intermediates is essential for bipolar chromosome segregation. Here, we show that the specialized chromosome segregation patterns of meiosis ...and mitosis, which require the coordination of recombination with cell-cycle progression, are achieved by regulating the timing of activation of two crossover-promoting endonucleases. In yeast meiosis, Mus81-Mms4 and Yen1 are controlled by phosphorylation events that lead to their sequential activation. Mus81-Mms4 is hyperactivated by Cdc5-mediated phosphorylation in meiosis I, generating the crossovers necessary for chromosome segregation. Yen1 is also tightly regulated and is activated in meiosis II to resolve persistent Holliday junctions. In yeast and human mitotic cells, a similar regulatory network restrains these nuclease activities until mitosis, biasing the outcome of recombination toward noncrossover products while also ensuring the elimination of any persistent joint molecules. Mitotic regulation thereby facilitates chromosome segregation while limiting the potential for loss of heterozygosity and sister-chromatid exchanges.
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► Mus81-Mms4 and Yen1 endonucleases collaboratively resolve DNA joint molecules in meiosis ► Cdc5 activates Mus81-Mms4 during meiosis I through the phosphorylation of Mms4 ► Phosphorylation inhibits Yen1 until the onset of meiosis II ► Yen1/GEN1 and Mus81-Mms4/EME1 are specifically activated during M phase of mitosis
Endonucleases resolve DNA recombination intermediates at different stages to promote and prevent crossing over in meiosis and mitosis, respectively.
Influenza hemagglutinin membrane anchor Benton, Donald J.; Nans, Andrea; Calder, Lesley J. ...
Proceedings of the National Academy of Sciences - PNAS,
10/2018, Letnik:
115, Številka:
40
Journal Article
Recenzirano
Odprti dostop
Viruses with membranes fuse them with cellular membranes, to transfer their genomes into cells at the beginning of infection. For Influenza virus, the membrane glycoprotein involved in fusion is the ...hemagglutinin (HA), the 3D structure of which is known from X-ray crystallographic studies. The soluble ectodomain fragments used in these studies lacked the “membrane anchor” portion of the molecule. Since this region has a role in membrane fusion, we have determined its structure by analyzing the intact, full-length molecule in a detergent micelle, using cryo-EM. We have also compared the structures of full-length HA−detergent micelles with full-length HA−Fab complex detergent micelles, to describe an infectivity-neutralizing monoclonal Fab that binds near the ectodomain membrane anchor junction. We determine a high-resolution HA structure which compares favorably in detail with the structure of the ectodomain seen by X-ray crystallography; we detect, clearly, all five carbohydrate side chains of HA; and we find that the ectodomain is joined to the membrane anchor by flexible, eight-residue-long, linkers. The linkers extend into the detergent micelle to join a central triple-helical structure that is a major component of the membrane anchor.
Coronaviruses of bats and pangolins have been implicated in the origin and evolution of the pandemic SARS-CoV-2. We show that spikes from Guangdong Pangolin-CoVs, closely related to SARS-CoV-2, bind ...strongly to human and pangolin ACE2 receptors. We also report the cryo-EM structure of a Pangolin-CoV spike protein and show it adopts a fully-closed conformation and that, aside from the Receptor-Binding Domain, it resembles the spike of a bat coronavirus RaTG13 more than that of SARS-CoV-2.
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