I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[6386]
Open Access
Abstract: The catalase from Scytalidium thermophilum is a homotetramer containing a heme d in each active site. Although the enzyme has a classical monofunctional catalase fold, it also possesses oxidase activity towards a number of small organics, including catechol and phenol. In order to further investigate this, the crystal structure of the complex of the catalase with the classical catalase inhibitor 3-amino-1,2,4-triazole (3TR) was determined at 1.95 Å resolution. Surprisingly, no binding to the heme site was observed; instead, 3TR occupies a binding site corresponding to the NADPH-binding pocket in mammalian catalases at the entrance to a lateral channel leading to the heme. Kinetic analysis of site-directed mutants supports the assignment of this pocket as the binding site for oxidase substrates.
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Oct 2018
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[6386]
Open Access
Abstract: Copper amine oxidases (CuAOs) are metalloenzymes that reduce molecular oxygen to hydrogen peroxide during catalytic turnover of primary amines. In addition to Cu2+ in the active site, two peripheral calcium sites, ca. 32 Å from the active site, have roles in Escherichia coli amine oxidase (ECAO). The buried Ca2+ (Asp533, Leu534, Asp535, Asp678, Ala 679) is essential for full-length protein production, while the surface Ca2+ (Glu573, Tyr667, Asp670, Glu672) modulates biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. The mutation E573Q at the surface-site prevents calcium binding and TPQ biogenesis. However, TPQ biogenesis can be restored by a suppressor mutation (I342F) in the proposed oxygen delivery channel to the active site. While supporting TPQ biogenesis (ca. 60 % WTECAO TPQ), I342F/E573Q has almost no amine oxidase activity (ca. 4.6 % WTECAO activity). To understand how these long range mutations result in major effects on TPQ biogenesis and catalysis we employed UV-vis spectroscopy, steady-state kinetics, inhibition assays and X-ray crystallography. We show that the surface metal site controls the equilibrium (disproportionation) of the Cu2+-substrate reduced TPQ (TPQAMQ) Cu1+-TPQ semiquinone (TPQSQ) couple. Removal of the calcium ion from this site by chelation or mutagenesis shifts the equilibrium to the Cu2+-TPQAMQ or destabilizes the Cu1+-TPQSQ. Crystal structure analysis shows that TPQ biogenesis is stalled at deprotonation in the Cu2+-tyrosinate state. Our findings support WTECAO using the inner sphere electron transfer mechanism for oxygen reduction during catalysis, and whilst a Cu1+-tyrosyl radical intermediate is not essential for TPQ biogenesis, it is required for efficient biogenesis.
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Aug 2018
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I24-Microfocus Macromolecular Crystallography
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James I.
Robinson
,
Euan W.
Baxter
,
Robin L.
Owen
,
Maren
Thomsen
,
Darren C.
Tomlinson
,
Mark P.
Waterhouse
,
Stephanie J.
Win
,
Joanne E.
Nettleship
,
Christian
Tiede
,
Richard J.
Foster
,
Raymond J.
Owens
,
Colin W. G.
Fishwick
,
Sarah A.
Harris
,
Adrian
Goldman
,
Michael J.
Mcpherson
,
Ann W.
Morgan
Diamond Proposal Number(s):
[5969]
Abstract: Protein–protein interactions are essential for the control of cellular functions and are critical for regulation of the immune system. One example is the binding of Fc regions of IgG to the Fc gamma receptors (FcγRs). High sequence identity (98%) between the genes encoding FcγRIIIa (expressed on macrophages and natural killer cells) and FcγRIIIb (expressed on neutrophils) has prevented the development of monospecific agents against these therapeutic targets. We now report the identification of FcγRIIIa-specific artificial binding proteins called “Affimer” that block IgG binding and abrogate FcγRIIIa-mediated downstream effector functions in macrophages, namely TNF release and phagocytosis. Cocrystal structures and molecular dynamics simulations have revealed the structural basis of this specificity for two Affimer proteins: One binds directly to the Fc binding site, whereas the other acts allosterically.
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Jan 2018
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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David J.
Hughes
,
Christian
Tiede
,
Natalie
Penswick
,
Anna Ah-San
Tang
,
Chi H.
Trinh
,
Upasana
Mandal
,
Katarzyna Z.
Zajac
,
Thembaninkosi
Gaule
,
Gareth
Howell
,
Thomas A.
Edwards
,
Jianxin
Duan
,
Eric
Feyfant
,
Michael J.
Mcpherson
,
Darren C.
Tomlinson
,
Adrian
Whitehouse
Abstract: Because protein-protein interactions underpin most biological processes, developing tools that target them to understand their function or to inform the development of therapeutics is an important task. SUMOylation is the posttranslational covalent attachment of proteins in the SUMO family (SUMO-1, SUMO-2, or SUMO-3), and it regulates numerous cellular pathways. SUMOylated proteins are recognized by proteins with SUMO-interaction motifs (SIMs) that facilitate noncovalent interactions with SUMO. We describe the use of the Affimer system of peptide display for the rapid isolation of synthetic binding proteins that inhibit SUMO-dependent protein-protein interactions mediated by SIMs both in vitro and in cells. Crucially, these synthetic proteins did not prevent SUMO conjugation either in vitro or in cell-based systems, enabling the specific analysis of SUMO-mediated protein-protein interactions. Furthermore, through structural analysis and molecular modeling, we explored the molecular mechanisms that may underlie their specificity in interfering with either SUMO-1–mediated interactions or interactions mediated by either SUMO-2 or SUMO-3. Not only will these reagents enable investigation of the biological roles of SUMOylation, but the Affimer technology used to generate these synthetic binding proteins could also be exploited to design or validate reagents or therapeutics that target other protein-protein interactions.
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Nov 2017
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E. W
Baxter
,
J. I
Robinson
,
D. C
Tomlinson
,
R. J
Foster
,
R. L
Owen
,
S. J
Win
,
J. E
Nettleship
,
C
Tiede
,
J
Kankanala
,
R. J
Owens
,
C. W. G
Fishwick
,
M. J
Mcpherson
,
A. W
Morgan
Abstract: Protein-protein interactions are essential for the control of cellular functions and critical for regulation of the immune system. One example is the binding of Fc regions of Immunoglobulin G to their receptors (Fcγ Receptors). High sequence identity (98%) between the genes encoding FcγRIIIa and FcγRIIIb has led to the lack of specific agents against this important therapeutic target. We aimed to develop a novel drug development pipeline using artificial binding proteins called Adhirons both for the identification of novel therapeutics and to guide drug discovery through the identification of novel hot spots/ druggable surfaces on the receptor.
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Feb 2016
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I24-Microfocus Macromolecular Crystallography
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C.
Tiede
,
A. A. S.
Tang
,
S. E.
Deacon
,
U.
Mandal
,
J. E.
Nettleship
,
R. L.
Owen
,
S. E.
George
,
D. J.
Harrison
,
R. J.
Owens
,
D. C.
Tomlinson
,
M. J.
Mcpherson
Diamond Proposal Number(s):
[5968]
Open Access
Abstract: We have designed a novel non-antibody scaffold protein, termed Adhiron, based on a phytocystatin consensus sequence. The Adhiron scaffold shows high thermal stability (Tm ca. 101°C), and is expressed well in Escherichia coli. We have determined the X-ray crystal structure of the Adhiron scaffold to 1.75 Å resolution revealing a compact cystatin-like fold. We have constructed a phage-display library in this scaffold by insertion of two variable peptide regions. The library is of high quality and complexity comprising 1.3 × 1010 clones. To demonstrate library efficacy, we screened against the yeast Small Ubiquitin-like Modifier (SUMO). In selected clones, variable region 1 often contained sequences homologous to the known SUMO interactive motif (V/I-X-V/I-V/I). Four Adhirons were further characterised and displayed low nanomolar affinities and high specificity for yeast SUMO with essentially no cross-reactivity to human SUMO protein isoforms. We have identified binders against >100 target molecules to date including as examples, a fibroblast growth factor (FGF1), platelet endothelial cell adhesion molecule (PECAM-1; CD31), the SH2 domain Grb2 and a 12-aa peptide. Adhirons are highly stable and well expressed allowing highly specific binding reagents to be selected for use in molecular recognition applications
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Apr 2014
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[8367]
Abstract: Almost all monofunctional haem catalases contain a highly conserved core containing the active site, which is connected to the exterior of the enzyme by three channels. These channels have been identified as potential routes for substrate flow and product release. To further investigate the role of these molecular channels, a series of mutants of Scytalidium thermophilum catalase were generated. The three-dimensional structures of four catalase variants, N155A, V123A, V123C and V123T, have been determined at resolutions of 2.25, 1.93, 1.9 and 1.7?Å, respectively. The V123C variant contains a new covalent bond between the S atom of Cys123 and the imidazole ring of the essential His82. This variant enzyme has only residual catalase activity and contains haem b instead of the normal haem d. The H82A variant demonstrates low catalase and phenol oxidase activities (0.2 and 20% of those of recombinant wild-type catalase–phenol oxidase, respectively). The N155A and N155H variants exhibit 4.5 and 3% of the wild-type catalase activity and contain haem d, showing that Asn155 is essential for catalysis but is not required for the conversion of haem b to haem d. Structural analysis suggests that the cause of the effect of these mutations on catalysis is the disruption of the ability of dioxygen substrates to efficiently access the active site. Additional mutants have been characterized biochemically to further probe the roles of the different channels. Introducing smaller or polar side chains in place of Val123 reduces the catalase activity. The F160V, F161V and F168V mutants show a marked decrease in catalase activity but have a much lower effect on the phenol oxidase activity, despite containing substoichiometric amounts of haem
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Apr 2013
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I03-Macromolecular Crystallography
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Abstract: Scytalidium thermophilum produces a catalase with phenol oxidase activity (CATPO) that catalyses the decomposition of hydrogen peroxide into oxygen and water and also oxidizes various phenolic compounds. A codon-optimized catpo gene was cloned and expressed in Escherichia coli. The crystal structures of native and recombinant S. thermophilum CATPO and two variants, H82N and V123F, were determined at resolutions of 2.7, 1.4, 1.5 and 1.9 Å, respectively. The structure of CATPO reveals a homotetramer with 698 residues per subunit and with strong structural similarity to Penicillium vitale catalase. The haem component is cis-hydroxychlorin γ-spirolactone, which is rotated 180° with respect to small-subunit catalases. The haem-binding pocket contains two highly conserved water molecules on the distal side. The H82N mutation resulted in conversion of the native d-type haem to a b-type haem. Kinetic studies of the H82N and V123F mutants indicate that both activities are likely to be associated with the haem centre and suggest that the secondary oxidase activity may be a general feature of catalases in the absence of hydrogen peroxide.
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Mar 2013
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Gerard H. M.
Huysmans
,
Nathan
Chan
,
Jocelyn M.
Baldwin
,
Vincent L. G.
Postis
,
Svetomir B.
Tzokov
,
Sarah E.
Deacon
,
Sylvia Y. M.
Yao
,
James D.
Young
,
Michael J.
Mcpherson
,
Per A.
Bullough
,
Stephen A.
Baldwin
Abstract: Urea is exploited as a nitrogen source by bacteria, and its breakdown products, ammonia and bicarbonate, are employed to counteract stomach acidity in pathogens such as Helicobacter pylori. Uptake in the latter is mediated by UreI, a UAC (urea amide channel) family member. In the present paper, we describe the structure and function of UACBc, a homologue from Bacillus cereus. The purified channel was found to be permeable not only to urea, but also to other small amides. CD and IR spectroscopy revealed a structure comprising mainly ?-helices, oriented approximately perpendicular to the membrane. Consistent with this finding, site-directed fluorescent labelling indicated the presence of seven TM (transmembrane) helices, with a cytoplasmic C-terminus. In detergent, UACBc exists largely as a hexamer, as demonstrated by both cross-linking and size-exclusion chromatography. A 9 Å (1 Å=0.1 nm) resolution projection map obtained by cryo-electron microscopy of two-dimensional crystals shows that the six protomers are arranged in a planar hexameric ring. Each exhibits six density features attributable to TM helices, surrounding a putative central channel, while an additional helix is peripherally located. Bioinformatic analyses allowed individual TM regions to be tentatively assigned to the density features, with the resultant model enabling identification of residues likely to contribute to channel function.
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Jul 2012
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[302]
Abstract: A key question in the biological activation of oxygen is how the protein matrix regulates the delivery of oxygen to its site of activation. We are using Escherichia coli copper amine oxidase as a model system to investigate the roles played by both local active site residues as well as long range interactions in this process. We have generated active site mutants, as well as mutants in the putative oxygen delivery channel and characterised their affects on enzyme structure and catalysis.
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Jan 2011
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