I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14774]
Open Access
Abstract: 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.
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Aug 2019
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I02-Macromolecular Crystallography
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Open Access
Abstract: In the disease familial amyloidosis, Finnish type (FAF), also known as AGel amyloidosis (AGel), the mechanism by which point mutations in the calcium-regulated actin-severing protein gelsolin lead to furin cleavage is not understood in the intact protein. Here, we provide a structural and biochemical characterization of the FAF variants. X-ray crystallography structures of the FAF mutant gelsolins demonstrate that the mutations do not significantly disrupt the calcium-free conformations of gelsolin. Small-angle X-ray–scattering (SAXS) studies indicate that the FAF calcium-binding site mutants are slower to activate, whereas G167R is as efficient as the wild type. Actin-regulating studies of the gelsolins at the furin cleavage pH (6.5) show that the mutant gelsolins are functional, suggesting that they also adopt relatively normal active conformations. Deletion of gelsolin domains leads to sensitization to furin cleavage, and nanobody-binding protects against furin cleavage. These data indicate instability in the second domain of gelsolin (G2), since loss or gain of G2-stabilizing interactions impacts the efficiency of cleavage by furin. To demonstrate this principle, we engineered non-FAF mutations in G3 that disrupt the G2-G3 interface in the calcium-activated structure. These mutants led to increased furin cleavage. We carried out molecular dynamics (MD) simulations on the FAF and non-FAF mutant G2-G3 fragments of gelsolin. All mutants showed an increase in the distance between the center of masses of the 2 domains (G2 and G3). Since G3 covers the furin cleavage site on G2 in calcium-activated gelsolin, this suggests that destabilization of this interface is a critical step in cleavage.
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Jun 2019
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[14856]
Abstract: Characterizing the genome of mature virions is pivotal to understanding the highly dynamic processes of virus assembly and infection. Owing to the different cellular fates of DNA and RNA, the life cycles of double-stranded (ds)DNA and dsRNA viruses are dissimilar. In terms of nucleic acid packing, dsDNA viruses, which lack genome segmentation and intra-capsid transcriptional machinery, predominantly display single-spooled genome organizations1,2,3,4,5,6,7,8. Because the release of dsRNA into the cytoplasm triggers host defence mechanisms9, dsRNA viruses retain their genomes within a core particle that contains the enzymes required for RNA replication and transcription10,11,12. The genomes of dsRNA viruses vary greatly in the degree of segmentation. In members of the Reoviridae family, genomes consist of 10–12 segments and exhibit a non-spooled arrangement mediated by RNA-dependent RNA polymerases11,12,13,14. However, whether this arrangement is a general feature of dsRNA viruses remains unknown. Here, using cryo-electron microscopy to resolve the dsRNA genome structure of the tri-segmented bacteriophage ɸ6 of the Cystoviridae family, we show that dsRNA viruses can adopt a dsDNA-like single-spooled genome organization. We find that in this group of viruses, RNA-dependent RNA polymerases do not direct genome ordering, and the dsRNA can adopt multiple conformations. We build a model that encompasses 90% of the genome, and use this to quantify variation in the packing density and to characterize the different liquid crystalline geometries that are exhibited by the tightly compacted nucleic acid. Our results demonstrate that the canonical model for the packing of dsDNA can be extended to dsRNA viruses.
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May 2019
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I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
Krios I-Titan Krios I at Diamond
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Kamel
El Omari
,
Sai
Li
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Abhay
Kotecha
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Thomas S.
Walter
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Eduardo A.
Bignon
,
Karl
Harlos
,
Pentti
Somerharju
,
Felix
De Haas
,
Daniel K.
Clare
,
Mika
Molin
,
Felipe
Hurtado
,
Mengqiu
Li
,
Jonathan
Grimes
,
Dennis H.
Bamford
,
Nicole D.
Tischler
,
Juha T.
Huiskonen
,
Dave I.
Stuart
,
Elina
Roine
Diamond Proposal Number(s):
[10627]
Open Access
Abstract: Lipid membrane fusion is an essential function in many biological processes. Detailed mechanisms of membrane fusion and the protein structures involved have been mainly studied in eukaryotic systems, whereas very little is known about membrane fusion in prokaryotes. Haloarchaeal pleomorphic viruses (HRPVs) have a membrane envelope decorated with spikes that are presumed to be responsible for host attachment and membrane fusion. Here we determine atomic structures of the ectodomains of the 57-kDa spike protein VP5 from two related HRPVs revealing a previously unreported V-shaped fold. By Volta phase plate cryo-electron tomography we show that VP5 is monomeric on the viral surface, and we establish the orientation of the molecules with respect to the viral membrane. We also show that the viral membrane fuses with the host cytoplasmic membrane in a process mediated by VP5. This sheds light on protein structures involved in prokaryotic membrane fusion.
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Feb 2019
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14744]
Abstract: 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, tumour 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 3.95Å. 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 disulphide bond that connects equivalent cysteine residues of the parallel helices in the leucine zipper region. This disulphide bond in the hydrophobic core of the bZIP may increase the stability of the homodimer under oxidising conditions. Moreover, sequence alignment of bZIP sequences from chicken, human and mouse reveals only members of the CREB3 subfamily contain this cysteine residue, indicating it could act as a redox‐sensor. Taken together, these results suggest activity of these transcription factors may be redox‐regulated and they may be activated in response to oxidative stress.
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Jan 2019
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I02-Macromolecular Crystallography
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Max
Renner
,
Aleksandra
Flanagan
,
Wanwisa
Dejnirattisai
,
Chunya
Puttikhunt
,
Watchara
Kasinrerk
,
Piyada
Supasa
,
Wiyada
Wongwiwat
,
Kriangkrai
Chawansuntati
,
Thaneeya
Duangchinda
,
Alison
Cowper
,
Claire M.
Midgley
,
Prida
Malasit
,
Juha T.
Huiskonen
,
Juthathip
Mongkolsapaya
,
Gavin R.
Screaton
,
Jonathan M.
Grimes
Diamond Proposal Number(s):
[8423]
Abstract: 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.
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Oct 2018
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Open Access
Abstract: Mononegaviruses, such as Ebola virus, encode an L (large) protein that bears all the catalytic activities for replication/transcription and RNA capping. The C-terminal conserved region VI (CRVI) of L protein contains a K-D-K-E catalytic tetrad typical for 2’O methyltransferases (MTase). In mononegaviruses, cap-MTase activities have been involved in the 2’O methylation and N7 methylation of the RNA cap structure. These activities play a critical role in the viral life cycle as N7 methylation ensures efficient viral mRNA translation and 2’O methylation hampers the detection of viral RNA by the host innate immunity. The functional characterization of the MTase+CTD domain of Sudan ebolavirus (SUDV) revealed cap-independent methyltransferase activities targeting internal adenosine residues. Besides this, the MTase+CTD also methylates, the N7 position of the cap guanosine and the 2’O position of the n1 guanosine provided that the RNA is sufficiently long. Altogether, these results suggest that the filovirus MTases evolved towards a dual activity with distinct substrate specificities. Whereas it has been well established that cap-dependent methylations promote protein translation and help to mimic host RNA, the characterization of an original cap-independent methylation opens new research opportunities to elucidate the role of RNA internal methylations in the viral replication.
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Sep 2018
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[10627]
Open Access
Abstract: CarD is an essential global transcription regulator from Mycobacterium tuberculosis (Mtb) that binds RNA polymerase and activates transcription by stabilizing the transcription initiation complex. Available crystal structures have captured two distinct, monomeric and domain-swapped homodimeric, oligomeric states of CarD. However, the actual oligomeric state of CarD in solution and its biological relevance has remained unclear. Here, we confirm the presence of the homodimeric state of CarD in solution by using synchrotron-based small-angle X-ray scattering. Furthermore, by using biochemical and biophysical experiments, in addition to mass-spectrometry, transmission electron microscopy, and confocal imaging, we show that CarD is the first soluble cytosolic protein in Mtb which displays the tendency to form amyloid-like fibrils both in vitro as well as in vivo. We demonstrate that the deletion of the fourteen N-terminal residues involved in domain-swapping hampers amyloid formation, thus, suggesting that domain-swapping is crucial in amyloidogenesis. The discovery of the amyloidogenic property of an essential cytosolic global transcription regulator, CarD, in a pathogenic bacteria will further open up new frontiers in research.
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Jul 2018
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8423]
Open Access
Abstract: 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.
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Jun 2018
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Helen M. E.
Duyvesteyn
,
Helen M.
Ginn
,
Maija K.
Pietila
,
Armin
Wagner
,
Johan
Hattne
,
Jonathan M.
Grimes
,
Elina
Hirvonen
,
Gwyndaf
Evans
,
Marie-laure
Parsy
,
Nicholas K.
Sauter
,
Aaron S.
Brewster
,
Juha
Huiskonen
,
David I.
Stuart
,
Geoff
Sutton
,
Dennis H.
Bamford
Open Access
Abstract: Viruses are a significant threat to both human health and the economy, and there is an urgent need for novel anti-viral drugs and vaccines. High-resolution viral structures inform our understanding of the virosphere, and inspire novel therapies. Here we present a method of obtaining such structural information that avoids potentially disruptive handling, by collecting diffraction data from intact infected cells. We identify a suitable combination of cell type and virus to accumulate particles in the cells, establish a suitable time point where most cells contain virus condensates and use electron microscopy to demonstrate that these are ordered crystalline arrays of empty capsids. We then use an X-ray free electron laser to provide extremely bright illumination of sub-micron intracellular condensates of bacteriophage phiX174 inside living Escherichia coli at room temperature. We have been able to collect low resolution diffraction data. Despite the limited resolution and completeness of these initial data, due to a far from optimal experimental setup, we have used novel methodology to determine a putative space group, unit cell dimensions, particle packing and likely maturation state of the particles.
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Feb 2018
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