I20-Scanning-X-ray spectroscopy (XAS/XES)
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Diamond Proposal Number(s):
[17053]
Abstract: Background: Copper toxicity and hepatic copper accumulation pose a serious risk to ruminant health and production. Differences in the copper-handling mechanisms of cattle and sheep have been noted, not only in comparison to each other, but also in comparison to ‘copper-tolerant’ monogastric species. Ruminants appear less able to cope with rising liver copper concentration than monogastric counterparts, with sheep in general less able to cope with elevated copper intake than cattle. Methods: X-ray absorption spectroscopy (XAS) was used to investigate the differences between the livers of these species at high copper status. Results: The X-ray absorption fine structure (XAFS) and X-ray absorption near edge structure (XANES) spectra indicated that the hepatic copper compound is most likely to be bound to metallothionein; consistent with monogastric species. Conclusion: Although, most likely stored as copper-metallothionein, there may be a role for glutathione as a short-term, intermediate copper buffer which may have more relevance to sheep than cattle. The potential that thiomolybdate bound copper can be stored in the liver could not be ruled out.
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Dec 2021
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I03-Macromolecular Crystallography
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Open Access
Abstract: Programmed –1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein, a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we present the X-ray crystal structure of 2A and show that it selectively binds to a pseudoknot-like conformation of the PRF stimulatory RNA element in the viral genome. Using optical tweezers, we demonstrate that 2A stabilises this RNA element, likely explaining the increase in PRF efficiency in the presence of 2A. Next, we demonstrate a strong interaction between 2A and the small ribosomal subunit and present a cryo-EM structure of 2A bound to initiated 70S ribosomes. Multiple copies of 2A bind to the 16S rRNA where they may compete for binding with initiation and elongation factors. Together, these results define the structural basis for RNA recognition by 2A, show how 2A-mediated stabilisation of an RNA pseudoknot promotes PRF, and reveal how 2A accumulation may shut down translation during virus infection.
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Dec 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[18548]
Open Access
Abstract: Viral diseases pose major threats to humans and other animals, including the billions of chickens that are an important food source as well as a public health concern due to zoonotic pathogens. Unlike humans and other typical mammals, the major histocompatibility complex (MHC) of chickens can confer decisive resistance or susceptibility to many viral diseases. An iconic example is Marek’s disease, caused by an oncogenic herpesvirus with over 100 genes. Classical MHC class I and class II molecules present antigenic peptides to T lymphocytes, and it has been hard to understand how such MHC molecules could be involved in susceptibility to Marek’s disease, given the potential number of peptides from over 100 genes. We used a new in vitro infection system and immunopeptidomics to determine peptide motifs for the 2 class II molecules expressed by the MHC haplotype B2, which is known to confer resistance to Marek’s disease. Surprisingly, we found that the vast majority of viral peptide epitopes presented by chicken class II molecules arise from only 4 viral genes, nearly all having the peptide motif for BL2*02, the dominantly expressed class II molecule in chickens. We expressed BL2*02 linked to several Marek’s disease virus (MDV) peptides and determined one X-ray crystal structure, showing how a single small amino acid in the binding site causes a crinkle in the peptide, leading to a core binding peptide of 10 amino acids, compared to the 9 amino acids in all other reported class II molecules. The limited number of potential T cell epitopes from such a complex virus can explain the differential MHC-determined resistance to MDV, but raises questions of mechanism and opportunities for vaccine targets in this important food species, as well as providing a basis for understanding class II molecules in other species including humans.
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Apr 2021
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Open Access
Abstract: For two decades, the genus pestivirus has been expanding and the host range now extends to rodents, bats and marine mammals. In this review, we focus on one of the most diverse pestiviruses, atypical porcine pestivirus or pestivirus K, comparing its special traits to what is already known at the structural and functional level from other pestiviruses.
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Apr 2021
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Nur Zazarina
Ramly
,
Samuel R.
Dix
,
Sergey N.
Ruzheinikov
,
Svetlana E.
Sedelnikova
,
Patrick J.
Baker
,
Yock-Ping
Chow
,
Fiona M.
Tomley
,
Damer P.
Blake
,
Kiew-Lian
Wan
,
Sheila
Nathan
,
David W.
Rice
Diamond Proposal Number(s):
[1218, 24447]
Open Access
Abstract: In infections by apicomplexan parasites including Plasmodium, Toxoplasma gondii, and Eimeria, host interactions are mediated by proteins including families of membrane-anchored cysteine-rich surface antigens (SAGs) and SAG-related sequences (SRS). Eimeria tenella causes caecal coccidiosis in chickens and has a SAG family with over 80 members making up 1% of the proteome. We have solved the structure of a representative E. tenella SAG, EtSAG19, revealing that, despite a low level of sequence similarity, the entire Eimeria SAG family is unified by its three-layer αβα fold which is related to that of the CAP superfamily. Furthermore, sequence comparisons show that the Eimeria SAG fold is conserved in surface antigens of the human coccidial parasite Cyclospora cayetanensis but this fold is unrelated to that of the SAGs/SRS proteins expressed in other apicomplexans including Plasmodium species and the cyst-forming coccidia Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti. However, despite having very different structures, Consurf analysis showed that Eimeria SAG and Toxoplasma SRS families each exhibit marked hotspots of sequence hypervariability that map to their surfaces distal to the membrane anchor. This suggests that the primary and convergent purpose of the different structures is to provide a platform onto which sequence variability can be imposed.
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Mar 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Olga V.
Moroz
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Elena
Blagova
,
Edward
Taylor
,
Johan
Turkenburg
,
Lars K.
Skov
,
Garry P.
Gippert
,
Kirk M.
Schnorr
,
Li
Ming
,
Liu
Ye
,
Mikkel
Klausen
,
Marianne T.
Cohn
,
Esben G. W.
Schmidt
,
Søren
Nymand-Grarup
,
Gideon J.
Davies
,
Keith S.
Wilson
Diamond Proposal Number(s):
[13587, 7864]
Open Access
Abstract: Muramidases/lysozymes hydrolyse the peptidoglycan component of the bacterial cell wall. They are found in many of the glycoside hydrolase (GH) families. Family GH25 contains muramidases/lysozymes, known as CH type lysozymes, as they were initially discovered in the Chalaropsis species of fungus. The characterized enzymes from GH25 exhibit both β-1,4-N-acetyl- and β-1,4-N,6-O-diacetylmuramidase activities, cleaving the β-1,4-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) moieties in the carbohydrate backbone of bacterial peptidoglycan. Here, a set of fungal GH25 muramidases were identified from a sequence search, cloned and expressed and screened for their ability to digest bacterial peptidoglycan, to be used in a commercial application in chicken feed. The screen identified the enzyme from Acremonium alcalophilum JCM 736 as a suitable candidate for this purpose and its relevant biochemical and biophysical and properties are described. We report the crystal structure of the A. alcalophilum enzyme at atomic, 0.78 Å resolution, together with that of its homologue from Trichobolus zukalii at 1.4 Å, and compare these with the structures of homologues. GH25 enzymes offer a new solution in animal feed applications such as for processing bacterial debris in the animal gut.
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Mar 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Lukasz F.
Sobala
,
Pearl Z.
Fernandes
,
Zalihe
Hakki
,
Andrew J.
Thompson
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Jonathon D.
Howe
,
Michelle
Hill
,
Nicole
Zitzmann
,
Scott
Davies
,
Zania
Stamataki
,
Terry D.
Butters
,
Dominic S.
Alonzi
,
Spencer J.
Williams
,
Gideon
Davies
Diamond Proposal Number(s):
[1221, 12587, 18598]
Open Access
Abstract: Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition, and function. N-linked glycans are synthesized from Glc3Man9GlcNAc2 precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-α-1,2-mannosidase (MANEA) is the sole endo-acting glycoside hydrolase involved in N-glycan trimming and is located within the Golgi, where it allows ER-escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur on substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. These structures have inspired the development of new inhibitors that disrupt host protein N-glycan processing of viral glycans and reduce the infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts aimed at developing broad-spectrum antiviral agents and help provide a more in-depth understanding of the biology of mammalian glycosylation.
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Nov 2020
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Sander
Herfst
,
Jie
Zhang
,
Mathilde
Richard
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Ryan
Mcbride
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Pascal
Lexmond
,
Theo M.
Bestebroer
,
Monique I. J.
Spronken
,
Dennis
De Meulder
,
Judith M.
Van Den Brand
,
Miruna E.
Rosu
,
Stephen R.
Martin
,
Steven J.
Gamblin
,
Xiaoli
Xiong
,
Wenjie
Peng
,
Rogier
Bodewes
,
Erhard
Van Der Vries
,
Albert D. M. E.
Osterhaus
,
James C.
Paulson
,
John J.
Skehel
,
Ron A. M.
Fouchier
Diamond Proposal Number(s):
[9826, 13775]
Abstract: In 2014, an outbreak of avian A/H10N7 influenza virus occurred among seals along North-European coastal waters, significantly impacting seal populations. Here, we examine the cross-species transmission and mammalian adaptation of this influenza A virus, revealing changes in the hemagglutinin surface protein that increase stability and receptor binding. The seal A/H10N7 virus was aerosol or respiratory droplet transmissible between ferrets. Compared with avian H10 hemagglutinin, seal H10 hemagglutinin showed stronger binding to the human-type sialic acid receptor, with preferential binding to α2,6-linked sialic acids on long extended branches. In X-ray structures, changes in the 220-loop of the receptor-binding pocket caused similar interactions with human receptor as seen for pandemic strains. Two substitutions made seal H10 hemagglutinin more stable than avian H10 hemagglutinin and similar to human hemagglutinin. Consequently, identification of avian-origin influenza viruses across mammals appears critical to detect influenza A viruses posing a major threat to humans and other mammals.
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Oct 2020
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I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[7254]
Open Access
Abstract: In this work, the effects of the protozoan Neospora caninum on the bioenergetics, chemical composition, and elemental content of human brain microvascular endothelial cells (hBMECs) were investigated. We showed that N. caninum can impair cell mitochondrial (Mt) function and causes an arrest in host cell cycling at S and G2 phases. These adverse effects were also associated with altered expression of genes involved in Mt energy metabolism, suggesting Mt dysfunction caused by N. caninum infection. Fourier Transform Infrared (FTIR) spectroscopy analysis of hBMECs revealed alterations in the FTIR bands as a function of infection, where infected cells showed alterations in the absorption bands of lipid (2924 cm−1), amide I protein (1649 cm−1), amide II protein (1537 cm−1), nucleic acids and carbohydrates (1092 cm−1, 1047 cm−1, and 939 cm−1). By using quantitative synchrotron radiation X-ray fluorescence (μSR-XRF) imaging and quantification of the trace elements Zn, Cu and Fe, we detected an increase in the levels of Zn and Cu from 3 to 24 h post infection (hpi) in infected cells compared to control cells, but there were no changes in the level of Fe. We also used Affymetrix array technology to investigate the global alteration in gene expression of hBMECs and rat brain microvascular endothelial cells (rBMVECs) in response to N. caninum infection at 24 hpi. The result of transcriptome profiling identified differentially expressed genes involved mainly in immune response, lipid metabolism and apoptosis. These data further our understanding of the molecular events that shape the interaction between N. caninum and blood-brain-barrier endothelial cells.
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Aug 2020
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12718]
Abstract: Trypanosoma are blood-borne parasites and are the causative agents of neglected tropical diseases (NTDs) affecting both humans and animals. These parasites mainly rely on glycolysis for their energy production within the mammalian host, which is why trypanosomal glycolytic enzymes have been pursued as interesting targets for the development of trypanocidal drugs. The structure–function relationships of pyruvate kinases (PYKs) from trypanosomatids (Trypanosoma and Leishmania) have been well-studied within this context. In this paper, we describe the structural and enzymatic characterization of PYK from T. congolense (TcoPYK), the main causative agent of Animal African Trypanosomosis (AAT), by employing a combination of enzymatic assays, thermal unfolding studies and X-ray crystallography.
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Mar 2020
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