I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Hannah R.
Adams
,
Dimitri A.
Svistunenko
,
Michael T.
Wilson
,
Sotaro
Fujii
,
Richard W.
Strange
,
Zoe A.
Hardy
,
Priscilla A.
Vazquez
,
Tyler
Dabritz
,
Gabriel J.
Streblow
,
Colin R.
Andrew
,
Michael A.
Hough
Diamond Proposal Number(s):
[13467, 18565, 25108]
Open Access
Abstract: The structural basis by which gas-binding heme proteins control their interactions with NO, CO, and O2, is fundamental to enzymology, biotechnology and human health. Cytochromes c´ (cyts c´) are a group of putative NO-binding heme proteins that fall into two families: the well characterised four alpha helix bundle fold (cyts c´-α) and an unrelated family with a largely beta sheet fold (cyts c´-β) resembling that of cytochromes P460. A recent structure of cyt c´-β from Methylococcus capsulatus Bath (McCP-β) revealed two heme pocket phenylalanine residues (Phe 32 and Phe 61) positioned near the distal gas binding site. This feature, dubbed the “Phe cap”, is highly conserved within the sequences of other cyts c´-β, but is absent in their close homologues, the hydroxylamine oxidizing cytochromes P460, although some do contain a single Phe residue. Here we report an integrated structural, spectroscopic, and kinetic characterization of McCP-β complexes with diatomic gases, focusing on the interaction of the Phe cap with NO and CO. Significantly, crystallographic and resonance Raman data show that orientation of the electron rich aromatic ring face of Phe 32 towards distally-bound NO or CO is associated with weakened backbonding and higher off rates. Moreover, we propose that an aromatic quadrupole also contributes to the unusually weak backbonding reported for some heme-based gas sensors, including the mammalian NO-sensor, soluble guanylate cyclase (sGC). Collectively, this study sheds light on the influence of highly conserved distal Phe residues on heme-gas complexes of cytochrome c’−β, including the potential for aromatic quadrupoles to modulate NO and CO binding in other heme proteins.
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Apr 2023
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I02-Macromolecular Crystallography
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Christos
Stergiou
,
Rhys
Williams
,
Jennifer R.
Fleming
,
Vasiliki
Zouvelou
,
Elpinickie
Ninou
,
Francesca
Andreetta
,
Elena
Rinaldi
,
Ornella
Simoncini
,
Renato
Mantegazza
,
Julius
Bogomolovas
,
John
Tzartos
,
Siegfried
Labeit
,
Olga
Mayans
,
Socrates
Tzartos
Diamond Proposal Number(s):
[8997]
Open Access
Abstract: Myasthenia gravis (MG) is an autoimmune disease caused by antibodies targeting the neuromuscular junction (NJ) of skeletal muscles. The major MG autoantigen is nicotinic acetylcholine receptor. Other autoantigens at the NJ include MuSK, LRP4 and agrin. Autoantibodies to the intra-sarcomeric striated muscle-specific gigantic protein titin, although not directed to the NJ, are invaluable biomarkers for thymoma and MG disease severity. Thymus and thymoma are critical in MG mechanisms and management. Titin autoantibodies bind to a 30 KDa titin segment, the main immunogenic region (MIR), consisting of an Ig-FnIII-FnIII 3-domain tandem, termed I109-I111. In this work, we further resolved the localization of titin epitope(s) to facilitate the development of more specific anti-titin diagnostics. For this, we expressed protein samples corresponding to 8 MIR and non-MIR titin fragments and tested 77 anti-titin sera for antibody binding using ELISA, competition experiments and Western blots. All anti-MIR antibodies were bound exclusively to the central MIR domain, I110, and to its containing titin segments. Most antibodies were bound also to SDS-denatured I110 on Western blots, suggesting that their epitope(s) are non-conformational. No significant difference was observed between thymoma and non-thymoma patients or between early- and late-onset MG. In addition, atomic 3D-structures of the MIR and its subcomponents were elucidated using X-ray crystallography. These immunological and structural data will allow further studies into the atomic determinants underlying titin-based autoimmunity, improved diagnostics and how to eventually treat titin autoimmunity associated co-morbidities.
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Feb 2023
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7707]
Open Access
Abstract: The linear ubiquitin chain assembly complex synthesises linear Ub chains which constitute a binding and activation platform for components of the TNF signalling pathway. One of the components of LUBAC is the ubiquitin ligase HOIL-1 which has been shown to generate oxyester linkages on several proteins and on linear polysaccharides. We show that HOIL-1 activity requires linear tetra-Ub binding which enables HOIL-1 to mono-ubiquitylate linear Ub chains and polysaccharides. Furthermore, we describe the crystal structure of a C-terminal tandem domain construct of HOIL-1 comprising the IBR and RING2 domains. Interestingly, the structure reveals a unique bi-nuclear Zn-cluster which substitutes the second zinc finger of the canonical RING2 fold. We identify the C-terminal histidine of this bi-nuclear Zn-cluster as the catalytic base required for the ubiquitylation activity of HOIL-1. Our study suggests that the unique zinc-coordinating architecture of RING2 provides a binding platform for ubiquitylation targets.
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Jan 2023
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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William M.
Dawson
,
Kathryn L.
Shelley
,
Jordan M.
Fletcher
,
D. Arne
Scott
,
Lucia
Lombardi
,
Guto G.
Rhys
,
Tania J.
Lagambina
,
Ulrike
Obst
,
Antony J.
Burton
,
Jessica A.
Cross
,
George
Davies
,
Freddie J. O.
Martin
,
Francis J.
Wiseman
,
R. Leo
Brady
,
David
Tew
,
Christopher W.
Wood
,
Derek N.
Woolfson
Diamond Proposal Number(s):
[12342, 23269]
Open Access
Abstract: Differential sensing attempts to mimic the mammalian senses of smell and taste to identify analytes and complex mixtures. In place of hundreds of complex, membrane-bound G-protein coupled receptors, differential sensors employ arrays of small molecules. Here we show that arrays of computationally designed de novo peptides provide alternative synthetic receptors for differential sensing. We use self-assembling α-helical barrels (αHBs) with central channels that can be altered predictably to vary their sizes, shapes and chemistries. The channels accommodate environment-sensitive dyes that fluoresce upon binding. Challenging arrays of dye-loaded barrels with analytes causes differential fluorophore displacement. The resulting fluorimetric fingerprints are used to train machine-learning models that relate the patterns to the analytes. We show that this system discriminates between a range of biomolecules, drink, and diagnostically relevant biological samples. As αHBs are robust and chemically diverse, the system has potential to sense many analytes in various settings.
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Jan 2023
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B21-High Throughput SAXS
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Laura C.
Clark
,
Kate E.
Atkin
,
Fiona
Whelan
,
Andrew S.
Brentnall
,
Gemma
Harris
,
Aisling M.
Towell
,
Johan P.
Turkenburg
,
Yan
Liu
,
Ten
Feizi
,
Samuel C.
Griffiths
,
Joan A.
Geoghegan
,
Jennifer R.
Potts
Diamond Proposal Number(s):
[7864, 18598]
Open Access
Abstract: Staphylococcus aureus and Staphylococcus epidermidis are frequently associated with medical device infections that involve establishment of a bacterial biofilm on the device surface. Staphylococcal surface proteins Aap, SasG and Pls are members of the Periscope Protein class and have been implicated in biofilm formation and host colonisation; they comprise a repetitive region (“B region”) and an N-terminal host colonisation domain within the “A region”, predicted to be a lectin domain. Repetitive E-G5 domains (as found in Aap, SasG and Pls) form elongated ‘stalks’ that would vary in length with repeat number, resulting in projection of the N-terminal A domain variable distances from the bacterial cell surface. Here, we present the structures of the lectin domains within A regions of SasG, Aap and Pls and a structure of the Aap lectin domain attached to contiguous E-G5 repeats, suggesting the lectin domains will sit at the tip of the variable length rod. We demonstrate that these isolated domains (Aap, SasG) are sufficient to bind to human host desquamated nasal epithelial cells. Previously, proteolytic cleavage or a deletion within the A domain have been reported to induce biofilm formation; the structures suggest a potential link between these observations. Intriguingly, whilst the Aap, SasG and Pls lectin domains bind a metal ion, they lack the non-proline cis peptide bond thought to be key for carbohydrate binding by the lectin fold. This suggestion of non-canonical ligand binding should be a key consideration when investigating the host cell interactions of these bacterial surface proteins.
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Jan 2023
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9948, 13587]
Open Access
Abstract: Peptide transporters play important nutritional and cell signalling roles in Bacillus subtilis, which are pronounced during stationary phase adaptations and development. Three high-affinity ATP-binding cassette (ABC) family transporters are involved in peptide uptake – the oligopeptide permease (Opp), another peptide permease (App) and a less well-characterized dipeptide permease (Dpp). Here we report crystal structures of the extracellular substrate binding proteins, OppA and DppE, which serve the Opp and Dpp systems, respectively. The structure of OppA was determined in complex with endogenous peptides, modelled as Ser-Asn-Ser-Ser, and with the sporulation-promoting peptide Ser-Arg-Asn-Val-Thr, which bind with Kd values of 0.4 and 2 µM, respectively, as measured by isothermal titration calorimetry. Differential scanning fluorescence experiments with a wider panel of ligands showed that OppA has highest affinity for tetra- and penta-peptides. The structure of DppE revealed the unexpected presence of a murein tripeptide (MTP) ligand, l-Ala-d-Glu-meso-DAP, in the peptide binding groove. The mode of MTP binding in DppE is different to that observed in the murein peptide binding protein, MppA, from Escherichia coli, suggesting independent evolution of these proteins from an OppA-like precursor. The presence of MTP in DppE points to a role for Dpp in the uptake and recycling of cell wall peptides, a conclusion that is supported by analysis of the genomic context of dpp, which revealed adjacent genes encoding enzymes involved in muropeptide catabolism in a gene organization that is widely conserved in Firmicutes.
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Dec 2022
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Lorena
Zuliani-Alvarez
,
Morten L.
Govasli
,
Jane
Rasaiyaah
,
Chris
Monit
,
Stephen O.
Perry
,
Rebecca P.
Sumner
,
Simon
Mcalpine-Scott
,
Claire
Dickson
,
K. M.
Rifat Faysal
,
Laura
Hilditch
,
Richard J.
Miles
,
Frederic
Bibollet-Ruche
,
Beatrice H.
Hahn
,
Till
Boecking
,
Nikos
Pinotsis
,
Leo C.
James
,
David A.
Jacques
,
Greg J.
Towers
Diamond Proposal Number(s):
[8547, 11235]
Open Access
Abstract: Of the 13 known independent zoonoses of simian immunodeficiency viruses to humans, only one, leading to human immunodeficiency virus (HIV) type 1(M) has become pandemic, causing over 80 million human infections. To understand the specific features associated with pandemic human-to-human HIV spread, we compared replication of HIV-1(M) with non-pandemic HIV-(O) and HIV-2 strains in myeloid cell models. We found that non-pandemic HIV lineages replicate less well than HIV-1(M) owing to activation of cGAS and TRIM5-mediated antiviral responses. We applied phylogenetic and X-ray crystallography structural analyses to identify differences between pandemic and non-pandemic HIV capsids. We found that genetic reversal of two specific amino acid adaptations in HIV-1(M) enables activation of TRIM5, cGAS and innate immune responses. We propose a model in which the parental lineage of pandemic HIV-1(M) evolved a capsid that prevents cGAS and TRIM5 triggering, thereby allowing silent replication in myeloid cells. We hypothesize that this capsid adaptation promotes human-to-human spread through avoidance of innate immune response activation.
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Nov 2022
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9306]
Open Access
Abstract: JmjC (Jumonji-C) domain-containing 5 (JMJD5) plays important roles in circadian regulation in plants and humans and is involved in embryonic development and cell proliferation. JMJD5 is a 2-oxoglutarate (2OG) and Fe(II) dependent oxygenase of the JmjC subfamily, which includes histone Nε-methyl lysine-demethylases (KDMs) and hydroxylases catalysing formation of stable alcohol products. JMJD5 is reported to have KDM activity, but has been shown to catalyse C-3 hydroxylation of arginine residues in sequences from human regulator of chromosome condensation domain-containing protein 1 (RCCD1) and ribosomal protein S6 (RPS6) in vitro. We report crystallographic analyses of human JMJD5 complexed with 2OG analogues, including the widely used hypoxia mimic pyridine-2,4-dicarboxylate, both D- and L-enantiomers of the oncometabolite 2-hydroxyglutarate, and a cyclic N-hydroxyimide. The results support the assignment of JMJD5 as a protein hydroxylase and reveal JMJD5 has an unusually compact 2OG binding pocket suitable for exploitation in development of selective inhibitors. They will be useful in the development of chemical probes to investigate the physiologically relevant roles of JMJD5 in circadian rhythm and development and explore its potential as a medicinal chemistry target.
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Nov 2022
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18812]
Abstract: Fluorescent proteins have revolutionized cell biology and cell imaging through their use as genetically encoded tags. Structural biology has been pivotal in understanding how their unique fluorescent properties manifest through the formation of the chromophore and how the spectral properties are tuned through interaction networks. This knowledge has in turn led to the construction of novel variants with new and improved properties. Here we describe the process by which fluorescent protein structures are determined, starting from recombinant protein production to structure determination by molecular replacement. We also describe how to incorporate and determine the structures of proteins containing non-natural amino acids. Recent advances in protein engineering have led to reprogramming of the genetic code to allow incorporation of new chemistry at designed residue positions, with fluorescent proteins being at the forefront of structural studies in this area. The impact of such new chemistry on protein structure is still limited; the accumulation of more protein structures will undoubtedly improve our understanding and ability to engineer proteins with new chemical functionality.
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Sep 2022
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B21-High Throughput SAXS
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8423]
Open Access
Abstract: Sizzled (Szl) is both a secreted frizzled related protein (sFRP) and a naturally occurring inhibitor of the zinc metalloproteinase bone morphogenetic protein-1 (BMP-1), a key regulator of extracellular matrix assembly and growth factor activation. Here we present a new crystal structure for Szl which differs from that previously reported by a large scale (90°) hinge rotation between its cysteine-rich and netrin-like domains. We also present results of a molecular docking analysis showing interactions likely to be involved in the inhibition of BMP-1 activity by Szl. When compared with known structures of BMP-1 in complex with small molecule inhibitors, this reveals features that may be helpful in the design of new inhibitors to prevent the excessive accumulation of extracellular matrix that is the hallmark of fibrotic diseases.
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Sep 2022
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