Structure of the N-RNA/P interface indicates mode of L/P recruitment to the nucleocapsid of human metapneumovirus.

Human metapneumovirus (HMPV) is a major cause of respiratory illness in young children. The HMPV polymerase (L) binds an obligate cofactor, the phosphoprotein (P). During replication and transcription, the L/P complex traverses the viral RNA genome, which is encapsidated within nucleoproteins (N). An essential interaction between N and a C-terminal region of P tethers the L/P polymerase to the template. This N-P interaction is also involved in the formation of cytoplasmic viral factories in infected cells, called inclusion bodies. To define how the polymerase component P recognizes N-encapsidated RNA (N-RNA) we employed cryogenic electron microscopy (cryo-EM) and molecular dynamics simulations, coupled to activity assays and imaging of inclusion bodies in cells. We report a 2.9 Å resolution structure of a triple-complex between multimeric N, bound to both RNA and the C-terminal region of P. Furthermore, we also present cryo-EM structures of assembled N in different oligomeric states, highlighting the plasticity of N. Combined with our functional assays, these structural data delineate in molecular detail how P attaches to N-RNA whilst retaining substantial conformational dynamics. Moreover, the N-RNA-P triple complex structure provides a molecular blueprint for the design of therapeutics to potentially disrupt the attachment of L/P to its template.

Experimental phasing opportunities for macromolecular crystallography at very long wavelengths.

Despite recent advances in cryo-electron microscopy and artificial intelligence-based model predictions, a significant fraction of structure determinations by macromolecular crystallography still requires experimental phasing, usually by means of single-wavelength anomalous diffraction (SAD) techniques. Most synchrotron beamlines provide highly brilliant beams of X-rays of between 0.7 and 2 Å wavelength. Use of longer wavelengths to access the absorption edges of biologically important lighter atoms such as calcium, potassium, chlorine, sulfur and phosphorus for native-SAD phasing is attractive but technically highly challenging. The long-wavelength beamline I23 at Diamond Light Source overcomes these limitations and extends the accessible wavelength range to λ = 5.9 Å. Here we report 22 macromolecular structures solved in this extended wavelength range, using anomalous scattering from a range of elements which demonstrate the routine feasibility of lighter atom phasing. We suggest that, in light of its advantages, long-wavelength crystallography is a compelling option for experimental phasing.

Flavivirus maturation leads to the formation of an occupied lipid pocket in the surface glycoproteins.

Flaviviruses such as Dengue (DENV) or Zika virus (ZIKV) assemble into an immature form within the endoplasmatic reticulum (ER), and are then processed by furin protease in the trans-Golgi. To better grasp maturation, we carry out cryo-EM reconstructions of immature Spondweni virus (SPOV), a human flavivirus of the same serogroup as ZIKV. By employing asymmetric localised reconstruction we push the resolution to 3.8 Å, enabling us to refine an atomic model which includes the crucial furin protease recognition site and a conserved Histidine pH-sensor. For direct comparison, we also solve structures of the mature forms of SPONV and DENV to 2.6 Å and 3.1 Å, respectively. We identify an ordered lipid that is present in only the mature forms of ZIKV, SPOV, and DENV and can bind as a consequence of rearranging amphipathic stem-helices of E during maturation. We propose a structural role for the pocket and suggest it stabilizes mature E.

High-resolution crystal structure of arthropod Eiger TNF suggests a mode of receptor engagement and altered surface charge within endosomes.

The tumour necrosis factor alpha (TNFα) superfamily of proteins are critical in numerous biological processes, such as in development and immunity. Eiger is the sole TNFα member described in arthropods such as in the important model organism Drosophila. To date there are no structural data on any Eiger protein. Here we present the structure of the TNF domain of Eiger from the fall armyworm Spodoptera frugiperda (SfEiger) to 1.7 Å from a serendipitously obtained crystal without prior knowledge of the protein sequence. Our structure confirms that canonical trimerization is conserved from ancestral TNFs and points towards a mode of receptor engagement. Furthermore, we observe numerous surface histidines on SfEiger, potentially acting as pH switches following internalization into endosomes. Our data contributes to the genome annotation of S. frugiperda, a voracious agricultural pest, and can serve as a basis for future structure-function investigations of the TNF system in related arthropods such as Drosophila.

Diversity of oligomerization in Drosophila semaphorins suggests a mechanism of functional fine-tuning.

Semaphorin ligands and their plexin receptors are one of the major cell guidance factors that trigger localised changes in the cytoskeleton. Binding of semaphorin homodimer to plexin brings two plexins in close proximity which is a prerequisite for plexin signalling. This model appears to be too simplistic to explain the complexity and functional versatility of these molecules. Here, we determine crystal structures for all members of Drosophila class 1 and 2 semaphorins. Unlike previously reported semaphorin structures, Sema1a, Sema2a and Sema2b show stabilisation of sema domain dimer formation via a disulfide bond. Unexpectedly, our structural and biophysical data show Sema1b is a monomer suggesting that semaphorin function may not be restricted to dimers. We demonstrate that semaphorins can form heterodimers with members of the same semaphorin class. This heterodimerization provides a potential mechanism for cross-talk between different plexins and co-receptors to allow fine-tuning of cell signalling.

Insights from the crystal structure of the chicken CREB3 bZIP suggest that members of the CREB3 subfamily transcription factors may be activated in response to oxidative stress.

cAMP response element binding Protein 3 (CREB3) is an endoplasmic reticulum (ER) membrane-bound transcription factor, which belongs to the basic leucine zipper (bZIP) superfamily of eukaryotic transcription factors. CREB3 plays a role in the ER-stress induced unfolded protein response (UPR) and is a multifunctional cellular factor implicated in a number of biological processes including cell proliferation and migration, tumor suppression, and immune-related gene expression. To gain structural insights into the transcription factor, we determined the crystal structure of the conserved bZIP domain of chicken CREB3 (chCREB3) to a resolution of 3.95 Å. The X-ray structure provides evidence that chCREB3 can form a stable homodimer. The chCREB3 bZIP has a structured, pre-formed DNA binding region, even in the absence of DNA, a feature that could potentially enhance both the DNA binding specificity and affinity of chCREB3. Significantly, the homodimeric bZIP possesses an intermolecular disulfide bond that connects equivalent cysteine residues of the parallel helices in the leucine zipper region. This disulfide bond in the hydrophobic core of the bZIP may increase the stability of the homodimer under oxidizing conditions. Moreover, sequence alignment of bZIP sequences from chicken, human, and mouse reveals that only members of the CREB3 subfamily contain this cysteine residue, indicating that it could act as a redox-sensor. Taken together, these results suggest that the activity of these transcription factors may be redox-regulated and they may be activated in response to oxidative stress.

Characterization of a potent and highly unusual minimally enhancing antibody directed against dengue virus.

Dengue virus is a major pathogen, and severe infections can lead to life-threatening dengue hemorrhagic fever. Dengue virus exists as four serotypes, and dengue hemorrhagic fever is often associated with secondary heterologous infections. Antibody-dependent enhancement (ADE) may drive higher viral loads in these secondary infections and is purported to result from antibodies that recognize dengue virus but fail to fully neutralize it. Here we characterize two antibodies, 2C8 and 3H5, that bind to the envelope protein. Antibody 3H5 is highly unusual as it not only is potently neutralizing but also promotes little if any ADE, whereas antibody 2C8 has strong capacity to promote ADE. We show that 3H5 shows resilient binding in endosomal pH conditions and neutralizes at low occupancy. Immunocomplexes of 3H5 and dengue virus do not efficiently interact with Fcγ receptors, which we propose is due to the binding mode of 3H5 and constitutes the primary mechanism of how ADE is avoided.

A Mechanism for the Activation of the Influenza Virus Transcriptase.

Influenza virus RNA polymerase (FluPol), a heterotrimer composed of PB1, PB2, and PA subunits (P3 in influenza C), performs both transcription and replication of the viral RNA genome. For transcription, FluPol interacts with the C-terminal domain (CTD) of RNA polymerase II (Pol II), which enables FluPol to snatch capped RNA primers from nascent host RNAs. Here, we describe the co-crystal structure of influenza C virus polymerase (FluPolC) bound to a Ser5-phosphorylated CTD (pS5-CTD) peptide. The position of the CTD-binding site at the interface of PB1, P3, and the flexible PB2 C-terminal domains suggests that CTD binding stabilizes the transcription-competent conformation of FluPol. In agreement, both cap snatching and capped primer-dependent transcription initiation by FluPolC are enhanced in the presence of pS5-CTD. Mutations of amino acids in the CTD-binding site reduce viral mRNA synthesis. We propose a model for the activation of the influenza virus transcriptase through its association with pS5-CTD of Pol II.

Structural dissection of human metapneumovirus phosphoprotein using small angle x-ray scattering.

The phosphoprotein (P) is the main and essential cofactor of the RNA polymerase (L) of non-segmented, negative-strand RNA viruses. P positions the viral polymerase onto its nucleoprotein-RNA template and acts as a chaperone of the nucleoprotein (N), thereby preventing nonspecific encapsidation of cellular RNAs. The phosphoprotein of human metapneumovirus (HMPV) forms homotetramers composed of a stable oligomerization domain (Pcore) flanked by large intrinsically disordered regions (IDRs). Here we combined x-ray crystallography of Pcore with small angle x-ray scattering (SAXS)-based ensemble modeling of the full-length P protein and several of its fragments to provide a structural description of P that captures its dynamic character, and highlights the presence of varyingly stable structural elements within the IDRs. We discuss the implications of the structural properties of HMPV P for the assembly and functioning of the viral transcription/replication machinery.

The conserved protein Seb1 drives transcription termination by binding RNA polymerase II and nascent RNA.

Termination of RNA polymerase II (Pol II) transcription is an important step in the transcription cycle, which involves the dislodgement of polymerase from DNA, leading to release of a functional transcript. Recent studies have identified the key players required for this process and showed that a common feature of these proteins is a conserved domain that interacts with the phosphorylated C-terminus of Pol II (CTD-interacting domain, CID). However, the mechanism by which transcription termination is achieved is not understood. Using genome-wide methods, here we show that the fission yeast CID-protein Seb1 is essential for termination of protein-coding and non-coding genes through interaction with S2-phosphorylated Pol II and nascent RNA. Furthermore, we present the crystal structures of the Seb1 CTD- and RNA-binding modules. Unexpectedly, the latter reveals an intertwined two-domain arrangement of a canonical RRM and second domain. These results provide important insights into the mechanism underlying eukaryotic transcription termination.

Nucleocapsid assembly in pneumoviruses is regulated by conformational switching of the N protein.

Non-segmented, (-)RNA viruses cause serious human diseases. Human metapneumovirus (HMPV), an emerging pathogen of this order of viruses (Mononegavirales) is one of the main causes of respiratory tract illness in children. To help elucidate the assembly mechanism of the nucleocapsid (the viral RNA genome packaged by the nucleoprotein N) we present crystallographic structures of HMPV N in its assembled RNA-bound state and in a monomeric state, bound to the polymerase cofactor P. Our structures reveal molecular details of how P inhibits the self-assembly of N and how N transitions between the RNA-free and RNA-bound conformational state. Notably, we observe a role for the C-terminal extension of N in directly preventing premature uptake of RNA by folding into the RNA-binding cleft. Our structures suggest a common mechanism of how the growth of the nucleocapsid is orchestrated, and highlight an interaction site representing an important target for antivirals.

Averaging of viral envelope glycoprotein spikes from electron cryotomography reconstructions using Jsubtomo.

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein 'spikes' is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 Å and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

Structural insights into the human metapneumovirus glycoprotein ectodomain.

Human metapneumovirus is a major cause of respiratory tract infections worldwide. Previous reports have shown that the viral attachment glycoprotein (G) modulates innate and adaptive immune responses, leading to incomplete immunity and promoting reinfection. Using bioinformatics analyses, static light scattering, and small-angle X-ray scattering, we show that the extracellular region of G behaves as a heavily glycosylated, intrinsically disordered polymer. We discuss potential implications of these findings for the modulation of immune responses by G.

Drastic changes in conformational dynamics of the antiterminator M2-1 regulate transcription efficiency in Pneumovirinae.

The M2-1 protein of human metapneumovirus (HMPV) is a zinc-binding transcription antiterminator which is highly conserved among pneumoviruses. We report the structure of tetrameric HMPV M2-1. Each protomer features a N-terminal zinc finger domain and an α-helical tetramerization motif forming a rigid unit, followed by a flexible linker and an α-helical core domain. The tetramer is asymmetric, three of the protomers exhibiting a closed conformation, and one an open conformation. Molecular dynamics simulations and SAXS demonstrate a dynamic equilibrium between open and closed conformations in solution. Structures of adenosine monophosphate- and DNA- bound M2-1 establish the role of the zinc finger domain in base-specific recognition of RNA. Binding to 'gene end' RNA sequences stabilized the closed conformation of M2-1 leading to a drastic shift in the conformational landscape of M2-1. We propose a model for recognition of gene end signals and discuss the implications of these findings for transcriptional regulation in pneumoviruses.DOI: http://dx.doi.org/10.7554/eLife.02674.001.

A single molecule perspective on the functional diversity of in vitro evolved ß-glucuronidase.

The mechanisms that drive the evolution of new enzyme activity have been investigated by comparing the kinetics of wild-type and in vitro evolved ß-glucuronidase (GUS) at the single molecule level. Several hundred single GUS molecules were separated in large arrays of 62,500 ultrasmall reaction chambers etched into the surface of a fused silica slide to observe their individual substrate turnover rates in parallel by fluorescence microscopy. Individual GUS molecules feature long-lived but divergent activity states, and their mean activity is consistent with classic Michaelis-Menten kinetics. The large number of single molecule substrate turnover rates is representative of the activity distribution within an entire enzyme population. Partially evolved GUS displays a much broader activity distribution among individual enzyme molecules than wild-type GUS. The broader activity distribution indicates a functional division of work between individual molecules in a population of partially evolved enzymes that-as so-called generalists-are characterized by their promiscuous activity with many different substrates.

Structure and self-assembly of the calcium binding matrix protein of human metapneumovirus.

The matrix protein (M) of paramyxoviruses plays a key role in determining virion morphology by directing viral assembly and budding. Here, we report the crystal structure of the human metapneumovirus M at 2.8 Å resolution in its native dimeric state. The structure reveals the presence of a high-affinity Ca²âº binding site. Molecular dynamics simulations (MDS) predict a secondary lower-affinity site that correlates well with data from fluorescence-based thermal shift assays. By combining small-angle X-ray scattering with MDS and ensemble analysis, we captured the structure and dynamics of M in solution. Our analysis reveals a large positively charged patch on the protein surface that is involved in membrane interaction. Structural analysis of DOPC-induced polymerization of M into helical filaments using electron microscopy leads to a model of M self-assembly. The conservation of the Ca²âº binding sites suggests a role for calcium in the replication and morphogenesis of pneumoviruses.

Solution and crystallographic structures of the central region of the phosphoprotein from human metapneumovirus.

Human metapneumovirus (HMPV) of the family Paramyxoviridae is a major cause of respiratory illness worldwide. Phosphoproteins (P) from Paramyxoviridae are essential co-factors of the viral RNA polymerase that form tetramers and possess long intrinsically disordered regions (IDRs). We located the central region of HMPV P (P(ced)) which is involved in tetramerization using disorder analysis and modeled its 3D structure ab initio using Rosetta fold-and-dock. We characterized the solution-structure of P(ced) using small angle X-ray scattering (SAXS) and carried out direct fitting to the scattering data to filter out incorrect models. Molecular dynamics simulations (MDS) and ensemble optimization were employed to select correct models and capture the dynamic character of P(ced). Our analysis revealed that oligomerization involves a compact central core located between residues 169-194 (P(core)), that is surrounded by flexible regions with α-helical propensity. We crystallized this fragment and solved its structure at 3.1 Å resolution by molecular replacement, using the folded core from our SAXS-validated ab initio model. The RMSD between modeled and experimental tetramers is as low as 0.9 Å, demonstrating the accuracy of the approach. A comparison of the structure of HMPV P to existing mononegavirales P(ced) structures suggests that P(ced) evolved under weak selective pressure. Finally, we discuss the advantages of using SAXS in combination with ab initio modeling and MDS to solve the structure of small, homo-oligomeric protein complexes.