Structure and function of Plasmodium actin II in the parasite mosquito stages.

Actins are filament-forming, highly-conserved proteins in eukaryotes. They are involved in essential processes in the cytoplasm and also have nuclear functions. Malaria parasites (Plasmodium spp.) have two actin isoforms that differ from each other and from canonical actins in structure and filament-forming properties. Actin I has an essential role in motility and is fairly well characterized. The structure and function of actin II are not as well understood, but mutational analyses have revealed two essential functions in male gametogenesis and in the oocyst. Here, we present expression analysis, high-resolution filament structures, and biochemical characterization of Plasmodium actin II. We confirm expression in male gametocytes and zygotes and show that actin II is associated with the nucleus in both stages in filament-like structures. Unlike actin I, actin II readily forms long filaments in vitro, and near-atomic structures in the presence or absence of jasplakinolide reveal very similar structures. Small but significant differences compared to other actins in the openness and twist, the active site, the D-loop, and the plug region contribute to filament stability. The function of actin II was investigated through mutational analysis, suggesting that long and stable filaments are necessary for male gametogenesis, while a second function in the oocyst stage also requires fine-tuned regulation by methylation of histidine 73. Actin II polymerizes via the classical nucleation-elongation mechanism and has a critical concentration of ~0.1 µM at the steady-state, like actin I and canonical actins. Similarly to actin I, dimers are a stable form of actin II at equilibrium.

Electron cryotomography of SARS-CoV-2 virions reveals cylinder-shaped particles with a double layer RNP assembly.

SARS-CoV-2 is a lipid-enveloped Betacoronavirus and cause of the Covid-19 pandemic. To study the three-dimensional architecture of the virus, we perform electron cryotomography (cryo-ET) on SARS-Cov-2 virions and three variants revealing particles of regular cylindrical morphology. The ribonucleoprotein particles packaging the genome in the virion interior form a dense, double layer assembly with a cylindrical shape related to the overall particle morphology. This organisation suggests structural interactions important to virus assembly.

Cryomicroscopy reveals the structural basis for a flexible hinge motion in the immunoglobulin M pentamer.

Immunoglobulin M (IgM) is the most ancient of the five isotypes of immunoglobulin (Ig) molecules and serves as the first line of defence against pathogens. Here, we use cryo-EM to image the structure of the human full-length IgM pentamer, revealing antigen binding domains flexibly attached to the asymmetric and rigid core formed by the Cµ4 and Cµ3 constant regions and the J-chain. A hinge is located at the Cµ3/Cµ2 domain interface, allowing Fabs and Cµ2 to pivot as a unit both in-plane and out-of-plane. This motion is different from that observed in IgG and IgA, where the two Fab arms are able to swing independently. A biased orientation of one pair of Fab arms results from asymmetry in the constant domain (Cµ3) at the IgM subunit interacting most extensively with the J-chain. This may influence the multi-valent binding to surface-associated antigens and complement pathway activation. By comparison, the structure of the Fc fragment in the IgM monomer is similar to that of the pentamer, but is more dynamic in the Cµ4 domain.

Reversible structural changes in the influenza hemagglutinin precursor at membrane fusion pH.

The subunits of the influenza hemagglutinin (HA) trimer are synthesized as single-chain precursors (HA0s) that are proteolytically cleaved into the disulfide-linked polypeptides HA1 and HA2. Cleavage is required for activation of membrane fusion at low pH, which occurs at the beginning of infection following transfer of cell-surface-bound viruses into endosomes. Activation results in extensive changes in the conformation of cleaved HA. To establish the overall contribution of cleavage to the mechanism of HA-mediated membrane fusion, we used cryogenic electron microscopy (cryo-EM) to directly image HA0 at neutral and low pH. We found extensive pH-induced structural changes, some of which were similar to those described for intermediates in the refolding of cleaved HA at low pH. They involve a partial extension of the long central coiled coil formed by melting of the preexisting secondary structure, threading it between the membrane-distal domains, and subsequent refolding as extended helices. The fusion peptide, covalently linked at its N terminus, adopts an amphipathic helical conformation over part of its length and is repositioned and packed against a complementary surface groove of conserved residues. Furthermore, and in contrast to cleaved HA, the changes in HA0 structure at low pH are reversible on reincubation at neutral pH. We discuss the implications of covalently restricted HA0 refolding for the cleaved HA conformational changes that mediate membrane fusion and for the action of antiviral drug candidates and cross-reactive anti-HA antibodies that can block influenza infectivity.

High-resolution structures of malaria parasite actomyosin and actin filaments.

Malaria is responsible for half a million deaths annually and poses a huge economic burden on the developing world. The mosquito-borne parasites (Plasmodium spp.) that cause the disease depend upon an unconventional actomyosin motor for both gliding motility and host cell invasion. The motor system, often referred to as the glideosome complex, remains to be understood in molecular terms and is an attractive target for new drugs that might block the infection pathway. Here, we present the high-resolution structure of the actomyosin motor complex from Plasmodium falciparum. The complex includes the malaria parasite actin filament (PfAct1) complexed with the class XIV myosin motor (PfMyoA) and its two associated light-chains. The high-resolution core structure reveals the PfAct1:PfMyoA interface in atomic detail, while at lower-resolution, we visualize the PfMyoA light-chain binding region, including the essential light chain (PfELC) and the myosin tail interacting protein (PfMTIP). Finally, we report a bare PfAct1 filament structure at improved resolution.

In situ structure and organization of the influenza C virus surface glycoprotein.

The lipid-enveloped influenza C virus contains a single surface glycoprotein, the haemagglutinin-esterase-fusion (HEF) protein, that mediates receptor binding, receptor destruction, and membrane fusion at the low pH of the endosome. Here we apply electron cryotomography and subtomogram averaging to describe the structural basis for hexagonal lattice formation by HEF on the viral surface. The conformation of the glycoprotein in situ is distinct from the structure of the isolated trimeric ectodomain, showing that a splaying of the membrane distal domains is required to mediate contacts that form the lattice. The splaying of these domains is also coupled to changes in the structure of the stem region which is involved in membrane fusion, thereby linking HEF's membrane fusion conformation with its assembly on the virus surface. The glycoprotein lattice can form independent of other virion components but we show a major role for the matrix layer in particle formation.

Structure and binding properties of Pangolin-CoV spike glycoprotein inform the evolution of SARS-CoV-2.

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.

Influenza hemagglutinin membrane anchor.

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.

Three-Dimensional Architecture of the Human BRCA1-A Histone Deubiquitinase Core Complex.

BRCA1 is a tumor suppressor found to be mutated in hereditary breast and ovarian cancer and plays key roles in the maintenance of genomic stability by homologous recombination repair. It is recruited to damaged chromatin as a component of the BRCA1-A deubiquitinase, which cleaves K63-linked ubiquitin chains attached to histone H2A and H2AX. BRCA1-A contributes to checkpoint regulation, repair pathway choice, and HR repair efficiency through molecular mechanisms that remain largely obscure. The structure of an active core complex comprising two Abraxas/BRCC36/BRCC45/MERIT40 tetramers determined by negative-stain electron microscopy (EM) reveals a distorted V-shape architecture in which a dimer of Abraxas/BRCC36 heterodimers sits at the base, with BRCC45/Merit40 pairs occupying each arm. The location and ubiquitin-binding activity of BRCC45 suggest that it may provide accessory interactions with nucleosome-linked ubiquitin chains that contribute to their efficient processing. Our data also suggest how ataxia telangiectasia mutated (ATM)-dependent BRCA1 dimerization may stabilize self-association of the entire BRCA1-A complex.

Cryomicroscopy provides structural snapshots of influenza virus membrane fusion.

The lipid-enveloped influenza virus enters host cells during infection by binding cell-surface receptors and, after receptor-mediated endocytosis, fusing with the membrane of the endosome and delivering the viral genome and transcription machinery into the host cell. These events are mediated by the hemagglutinin (HA) surface glycoprotein. At the low pH of the endosome, an irreversible conformational change in the HA, including the exposure of the hydrophobic fusion peptide, activates membrane fusion. Here we used electron cryomicroscopy and cryotomography to image the fusion of influenza virus with target membranes at low pH. We visualized structural intermediates of HA and their interactions with membranes during the course of membrane fusion as well as ultrastructural changes in the virus that accompany membrane fusion. Our observations are relevant to a wide range of protein-mediated membrane-fusion processes and demonstrate how dynamic membrane events may be studied by cryomicroscopy.

Structures of complexes formed by H5 influenza hemagglutinin with a potent broadly neutralizing human monoclonal antibody.

H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection, in vitro and in vivo. As a consequence, this antibody may be suitable for anti-H5 therapy and as a component of stockpiles, together with other antiviral agents, for health authorities to use if an appropriate vaccine was not available. Our mutation and structural analyses indicate that the antibody recognizes a relatively conserved site near the membrane distal tip of HA, near to, but distinct from, the receptor-binding site. Our analyses also suggest that the mechanism of infectivity neutralization involves prevention of receptor recognition as a result of steric hindrance by the Fc part of the antibody. Structural analyses by EM indicate that three Fab fragments are bound to each HA trimer. The structure revealed by X-ray crystallography is of an HA monomer bound by one Fab. The monomer has some similarities to HA in the fusion pH conformation, and the monomer's formation, which results from the presence of isopropanol in the crystallization solvent, contributes to considerations of the process of change in conformation required for membrane fusion.

Structural studies of postentry restriction factors reveal antiparallel dimers that enable avid binding to the HIV-1 capsid lattice.

Restriction factors (RFs) form important components of host defenses to retroviral infection. The Fv1, Trim5α, and TrimCyp RFs contain N-terminal dimerization and C-terminal specificity domains that target assembled retroviral capsid (CA) proteins enclosing the viral core. However, the molecular detail of the interaction between RFs and their CA targets is unknown. Therefore, we have determined the crystal structure of the B-box and coiled-coil (BCC) region from Trim5α and used small-angle X-ray scattering to examine the solution structure of Trim5α BCC, the dimerization domain of Fv1 (Fv1Ntd), and the hybrid restriction factor Fv1Cyp comprising Fv1NtD fused to the HIV-1 binding protein Cyclophilin A (CypA). These data reveal that coiled-coil regions of Fv1 and Trim5α form extended antiparallel dimers. In Fv1Cyp, two CypA moieties are located at opposing ends, creating a molecule with a dumbbell appearance. In Trim5α, the B-boxes are located at either end of the coiled-coil, held in place by interactions with a helical motif from the L2 region of the opposing monomer. A comparative analysis of Fv1Cyp and CypA binding to a preformed HIV-1 CA lattice reveals how RF dimerization enhances the affinity of interaction through avidity effects. We conclude that the antiparallel organization of the NtD regions of Fv1 and Trim5α dimers correctly positions C-terminal specificity and N-terminal effector domains and facilitates stable binding to adjacent CA hexamers in viral cores.

Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation.

Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV_F occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV_F, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV_F at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule.

Cryomicroscopy of radiation sensitive specimens on unmodified graphene sheets: reduction of electron-optical effects of charging.

Images of radiation-sensitive specimens obtained by electron microscopy suffer a reduction in quality beyond that expected from radiation damage alone due to electron beam-induced charging or movement of the specimen. For biological specimens, charging and movement are most severe when they are suspended in an insulating layer of vitreous ice, which is otherwise optimal for preserving hydrated specimens in a near native state. We image biological specimens, including a single particle protein complex and a lipid-enveloped virus in thin, vitreous ice films over suspended sheets of unmodified graphene. We show that in such preparations, the charging of ice, as assessed by electron-optical perturbation of the imaging beam, is eliminated. We also use the same specimen supports to record high resolution images at liquid nitrogen temperature of monolayer paraffin crystals grown over graphene.

Distribution of surface glycoproteins on influenza A virus determined by electron cryotomography.

We use electron cryotomography to reconstruct virions of two influenza A H3N2 virus strains. The maps reveal the structure of the viral envelope containing hemagglutinin (HA) and neuraminidase (NA) glycoproteins and the virus interior containing a matrix layer and an assembly of ribonucleoprotein particles (RNPs) that package the genome. We build a structural model for the viral surface by locating copies of the X-ray structure of the HA ectodomain into density peaks on the virus surface. We calculate inter-glycoprotein distances and the fractional volume occupied by glycoproteins. The models suggest that for typical HA densities on virus, Fabs can bind to epitopes on the HA stem domain. The models also show how membrane curvature may influence the number of glycoproteins that can simultaneously interact with a target surface of receptors.

A human antibody recognizing a conserved epitope of H5 hemagglutinin broadly neutralizes highly pathogenic avian influenza H5N1 viruses.

Influenza A virus infection is a persistent threat to public health worldwide due to its ability to evade immune surveillance through rapid genetic drift and shift. Current vaccines against influenza A virus provide immunity to viral isolates that are similar to vaccine strains. High-affinity neutralizing antibodies against conserved epitopes could provide immunity to diverse influenza virus strains and protection against future pandemic viruses. In this study, by using a highly sensitive H5N1 pseudotype-based neutralization assay to screen human monoclonal antibodies produced by memory B cells from an H5N1-infected individual and molecular cloning techniques, we developed three fully human monoclonal antibodies. Among them, antibody 65C6 exhibited potent neutralization activity against all H5 clades and subclades except for subclade 7.2 and prophylactic and therapeutic efficacy against highly pathogenic avian influenza H5N1 viruses in mice. Studies on hemagglutinin (HA)-antibody complexes by electron microscopy and epitope mapping indicate that antibody 65C6 binds to a conformational epitope comprising amino acid residues at positions 118, 121, 161, 164, and 167 (according to mature H5 numbering) on the tip of the membrane-distal globular domain of HA. Thus, we conclude that antibody 65C6 recognizes a neutralization epitope in the globular head of HA that is conserved among almost all divergent H5N1 influenza stains.

A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins.

The isolation of broadly neutralizing antibodies against influenza A viruses has been a long-sought goal for therapeutic approaches and vaccine design. Using a single-cell culture method for screening large numbers of human plasma cells, we isolated a neutralizing monoclonal antibody that recognized the hemagglutinin (HA) glycoprotein of all 16 subtypes and neutralized both group 1 and group 2 influenza A viruses. Passive transfer of this antibody conferred protection to mice and ferrets. Complexes with HAs from the group 1 H1 and the group 2 H3 subtypes analyzed by x-ray crystallography showed that the antibody bound to a conserved epitope in the F subdomain. This antibody may be used for passive protection and to inform vaccine design because of its broad specificity and neutralization potency.

The Josephin domain determines the morphological and mechanical properties of ataxin-3 fibrils.

Fibrillar aggregation of the protein ataxin-3 is linked to the inherited neurodegenerative disorder Spinocerebellar ataxia type 3, a member of the polyQ expansion disease family. We previously reported that aggregation and stability of the nonpathological form of ataxin-3, carrying an unexpanded polyQ tract, are modulated by its N-terminal Josephin domain. It was also shown that expanded ataxin-3 aggregates via a two-stage mechanism initially involving Josephin self-association, followed by a polyQ-dependent step. Despite this recent progress, however, the exact mechanism of ataxin-3 fibrilization remains elusive. Here, we have used electron microscopy, atomic force microscopy, and other biophysical techniques to characterize the morphological and mechanical properties of nonexpanded ataxin-3 fibrils. By comparing aggregates of ataxin-3 and of the isolated Josephin domain, we show that the two proteins self-assemble into fibrils with markedly similar features over the temperature range 37-50°C. Estimates of persistence length and Young's modulus of the fibrils reveal a great flexibility. Our data indicate that, under physiological conditions, during early aggregation Josephin retains a nativelike secondary structure but loses its enzymatic activity. The results suggest a key role of Josephin in ataxin-3 fibrillar aggregation.

Functional interactions as a survival strategy against abnormal aggregation.

Protein aggregation is under intense scrutiny because of its role in human disease. Although increasing evidence indicates that protein native states are highly protected against aggregation, the specific protection mechanisms are poorly understood. Insight into such mechanisms can be gained through study of the relatively few proteins that aggregate under native conditions. Ataxin-3, the protein responsible for Spinocerebellar ataxia type 3, a polyglutamine expansion disease, represents one of such examples. Polyglutamine expansion is central for determining solubility and aggregation rates of ataxin-3, but these properties are profoundly modulated by its N-terminal Josephin domain. This work aims at identifying the regions that promote Josephin fibrillogenesis and rationalizing the mechanisms that protect Josephin and nonexpanded ataxin-3 from aberrant aggregation. Using different biophysical techniques, aggregation propensity predictions and rational design of amino acid substitutions, we show that Josephin has an intrinsic tendency to fibrillize under native conditions and that fibrillization is promoted by two solvent-exposed patches, which are also involved in recognition of natural substrates, such as ubiquitin. Indeed, designed mutations at these patches or substrate binding significantly reduce Josephin aggregation kinetics. Our results provide evidence that protein nonpathologic function can play an active role in preventing aberrant fibrillization and suggest the molecular mechanism whereby this occurs in ataxin-3.

Malaria parasite actin polymerization and filament structure.

A novel form of acto-myosin regulation has been proposed in which polymerization of new actin filaments regulates motility of parasites of the apicomplexan class of protozoa. In vivo and in vitro parasite F-actin is very short and unstable, but the structural basis and details of filament dynamics remain unknown. Here, we show that long actin filaments can be obtained by polymerizing unlabeled rabbit skeletal actin (RS-actin) onto both ends of the short rhodamine-phalloidin-stabilized Plasmodium falciparum actin I (Pf-actin) filaments. Following annealing, hybrid filaments of micron length and "zebra-striped" appearance are observed by fluorescence microscopy that are stable enough to move over myosin class II motors in a gliding filament assay. Using negative stain electron microscopy we find that pure Pf-actin stabilized by jasplakinolide (JAS) also forms long filaments, indistinguishable in length from RS-actin filaments, and long enough to be characterized structurally. To compare structures in near physiological conditions in aqueous solution we imaged Pf-actin and RS-actin filaments by atomic force microscopy (AFM). We found the monomer stacking to be distinctly different for Pf-actin compared with RS-actin, such that the pitch of the double helix of Pf-actin filaments was 10% larger. Our results can be explained by a rotational angle between subunits that is larger in the parasite compared with RS-actin. Modeling of the AFM data using high-resolution actin filament models supports our interpretation of the data. The structural differences reported here may be a consequence of weaker inter- and intra-strand contacts, and may be critical for differences in filament dynamics and for regulation of parasite motility.