I04-Macromolecular Crystallography
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Christopher T.
Lohans
,
David Y.
Wang
,
Christian
Jorgensen
,
Samuel T.
Cahill
,
Ian J.
Clifton
,
Michael A.
Mcdonough
,
Henry P.
Oswin
,
James
Spencer
,
Carmen
Domene
,
Timothy D. W.
Claridge
,
Jurgen
Brem
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[12346]
Abstract: The class D (OXA) serine β-lactamases are a major cause of resistance to β-lactam antibiotics. The class D enzymes are unique amongst β-lactamases because they have a carbamylated lysine that acts as a general acid/base in catalysis. Previous crystallographic studies led to the proposal that β-lactamase inhibitor avibactam targets OXA enzymes in part by promoting decarbamylation. Similarly, halide ions are proposed to inhibit OXA enzymes via decarbamylation. NMR analyses, in which the carbamylated lysines of OXA-10, -23 and -48 were 13C-labelled, indicate that reaction with avibactam does not ablate lysine carbamylation in solution. While halide ions did not decarbamylate the 13C-labelled OXA enzymes in the absence of substrate or inhibitor, avibactam-treated OXA enzymes were susceptible to decarbamylation mediated by halide ions, suggesting halide ions may inhibit OXA enzymes by promoting decarbamylation of acyl-enzyme complex. Crystal structures of the OXA-10 avibactam complex were obtained with bromide, iodide, and sodium ions bound between Trp-154 and Lys-70. Structures were also obtained wherein bromide and iodide ions occupy the position expected for the ‘hydrolytic water’ molecule. In contrast with some solution studies, Lys-70 was decarbamylated in these structures. These results reveal clear differences between crystallographic and solution studies on the interaction of class D β-lactamases with avibactam and halides, and demonstrate the utility of 13C-NMR for studying lysine carbamylation in solution.
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Jun 2017
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I24-Microfocus Macromolecular Crystallography
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Martine I.
Abboud
,
Philip
Hinchliffe
,
Jurgen
Brem
,
Robert
Macsics
,
Inga
Pfeffer
,
Anne
Makena
,
Klaus-daniel
Umland
,
Anna M.
Rydzik
,
Guo-bo
Li
,
James
Spencer
,
Timothy D. W.
Claridge
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[8922]
Abstract: Resistance to β-lactam antibiotics mediated by metallo-β-lactamases (MBLs) is a growing problem. We describe the use of protein-observe 19F-NMR (PrOF NMR) to study the dynamics of the São Paulo MBL (SPM-1) from β-lactam-resistant Pseudomonas aeruginosa. Cysteinyl variants on the α3 and L3 regions, which flank the di-ZnII active site, were selectively 19F-labeled using 3-bromo-1,1,1-trifluoroacetone. The PrOF NMR results reveal roles for the mobile α3 and L3 regions in the binding of both inhibitors and hydrolyzed β-lactam products to SPM-1. These results have implications for the mechanisms and inhibition of MBLs by β-lactams and non-β-lactams and illustrate the utility of PrOF NMR for efficiently analyzing metal chelation, identifying new binding modes, and studying protein binding from a mixture of equilibrating isomers.
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Mar 2017
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[12346]
Abstract: Bacterial production of β‐lactamases with carbapenemase activity is a global health threat. The active sites of class D carbapenemases such as OXA‐48, which is of major clinical importance, uniquely contain a carbamylated lysine residue which is essential for catalysis. Although there is significant interest in characterizing this post‐translational modification, and it is a promising inhibition target, protein carbamylation is challenging to monitor in solution. We report the use of 19F‐NMR spectroscopy to monitor the carbamylation state of 19F‐labelled OXA‐48. This method was used to investigate the interactions of OXA‐48 with clinically used serine β‐ lactamase inhibitors, including avibactam and vaborbactam. Crystallographic studies on 19F‐labelled OXA‐48 provide a structural rationale for the sensitivity of the 19F‐label to active site interactions. The overall results demonstrate the use of 19F‐NMR to monitor reversible covalent post‐translational modifications.
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Jul 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Hwanho
Choi
,
Adam P.
Hardy
,
Thomas M.
Leissing
,
Rasheduzzaman
Chowdhury
,
Yu
Nakashima
,
Wei
Ge
,
Marios
Markoulides
,
John S.
Scotti
,
Philip A.
Gerken
,
Helen
Thorbjornsrud
,
Dahye
Kang
,
Sungwoo
Hong
,
Joongoo
Lee
,
Michael A.
Mcdonough
,
Hwangseo
Park
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[18069]
Open Access
Abstract: Factor inhibiting hypoxia-inducible factor (FIH) is a 2-oxoglutarate-dependent protein hydroxylase that catalyses C3 hydroxylations of protein residues. We report FIH can accept (D)- and (L)-residues for hydroxylation. The substrate selectivity of FIH differs for (D) and (L) epimers, e.g., (D)- but not (L)-allylglycine, and conversely (L)- but not (D)-aspartate, undergo monohydroxylation, in the tested sequence context. The (L)-Leu-containing substrate undergoes FIH-catalysed monohydroxylation, whereas (D)-Leu unexpectedly undergoes dihydroxylation. Crystallographic, mass spectrometric, and DFT studies provide insights into the selectivity of FIH towards (L)- and (D)-residues. The results of this work expand the potential range of known substrates hydroxylated by isolated FIH and imply that it will be possible to generate FIH variants with altered selectivities.
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May 2020
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I04-Macromolecular Crystallography
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Laurens
Kruidenier
,
Chun-wa
Chung
,
Zhongjun
Cheng
,
John
Liddle
,
Kahing
Che
,
Gerard
Joberty
,
Marcus
Bantscheff
,
Chas
Bountra
,
Angela
Bridges
,
Hawa
Diallo
,
Dirk
Eberhard
,
Sue
Hutchinson
,
Emma
Jones
,
Roy
Katso
,
Melanie
Leveridge
,
Palwinder K.
Mander
,
Julie
Mosley
,
Cesar
Ramirez-molina
,
Paul
Rowland
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[7495]
Abstract: The jumonji (JMJ) family of histone demethylases are Fe2+- and ?-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes1, 2, 3, 4. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context5. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance2, 4, 6, as well as in development, physiology and disease3, 7. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX)8. The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.
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Aug 2012
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I24-Microfocus Macromolecular Crystallography
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Patrick
Rabe
,
John
Beale
,
Agata
Butryn
,
Pierre
Aller
,
Anna
Dirr
,
Pauline A.
Lang
,
Danny N.
Axford
,
Stephen
Carr
,
Thomas M.
Leissing
,
Michael A.
Mcdonough
,
Bradley
Davy
,
Ali
Ebrahim
,
Julien
Orlans
,
Selina L. S.
Storm
,
Allen M.
Orville
,
Christopher J.
Schofield
,
Robin L.
Owen
Diamond Proposal Number(s):
[19458]
Open Access
Abstract: Cryogenic X-ray diffraction is a powerful tool for crystallographic studies on enzymes including oxygenases and oxidases. Amongst the benefits that cryo-conditions (usually employing a nitrogen cryo-stream at 100 K) enable, is data collection of dioxygen-sensitive samples. Although not strictly anaerobic, at low temperatures the vitreous ice conditions severely restrict O2 diffusion into and/or through the protein crystal. Cryo-conditions limit chemical reactivity, including reactions that require significant conformational changes. By contrast, data collection at room temperature imposes fewer restrictions on diffusion and reactivity; room-temperature serial methods are thus becoming common at synchrotrons and XFELs. However, maintaining an anaerobic environment for dioxygen-dependent enzymes has not been explored for serial room-temperature data collection at synchrotron light sources. This work describes a methodology that employs an adaptation of the `sheet-on-sheet' sample mount, which is suitable for the low-dose room-temperature data collection of anaerobic samples at synchrotron light sources. The method is characterized by easy sample preparation in an anaerobic glovebox, gentle handling of crystals, low sample consumption and preservation of a localized anaerobic environment over the timescale of the experiment (<5 min). The utility of the method is highlighted by studies with three X-ray-radiation-sensitive Fe(II)-containing model enzymes: the 2-oxoglutarate-dependent L-arginine hydroxylase VioC and the DNA repair enzyme AlkB, as well as the oxidase isopenicillin N synthase (IPNS), which is involved in the biosynthesis of all penicillin and cephalosporin antibiotics.
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Sep 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[12346]
Open Access
Abstract: While the oxygen-dependent reversal of lysine Nɛ-methylation is well established, the existence of bona fide Nω-methylarginine demethylases (RDMs) is controversial. Lysine demethylation, as catalysed by two families of lysine demethylases (the flavin-dependent KDM1 enzymes and the 2-oxoglutarate- and oxygen-dependent JmjC KDMs, respectively), proceeds via oxidation of the N-methyl group, resulting in the release of formaldehyde. Here we report detailed biochemical studies clearly demonstrating that, in purified form, a subset of JmjC KDMs can also act as RDMs, both on histone and non-histone fragments, resulting in formaldehyde release. RDM catalysis is studied using peptides of wild-type sequences known to be arginine-methylated and sequences in which the KDM’s methylated target lysine is substituted for a methylated arginine. Notably, the preferred sequence requirements for KDM and RDM activity vary even with the same JmjC enzymes. The demonstration of RDM activity by isolated JmjC enzymes will stimulate efforts to detect biologically relevant RDM activity.
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Jun 2016
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I02-Macromolecular Crystallography
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Ming
Yang
,
Wei
Ge
,
Rasheduzzaman
Chowdhury
,
Timothy
Claridge
,
Holger
Kramer
,
Bernhard
Schmierer
,
Michael A.
Mcdonough
,
Lingzhi
Gong
,
Benedikt
Kessler
,
Peter
Ratcliffe
,
Mathew
Coleman
,
Christopher
Schofield
Abstract: Cytoskeleton, Post-translational Modification, Protein Stability, Protein Structure, Proteomics, Ankyrin Repeat Domain, AnkyrinR, Factor Inhibiting HIF, Hydroxylation, Hypoxia-inducible Factor
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Mar 2011
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Inga
Pfeffer
,
Lennart
Brewitz
,
Tobias
Krojer
,
Sacha A.
Jensen
,
Grazyna T.
Kochan
,
Nadia J.
Kershaw
,
Kirsty S.
Hewitson
,
Luke A.
Mcneill
,
Holger
Kramer
,
Martin
Münzel
,
Richard J.
Hopkinson
,
Udo
Oppermann
,
Penny A.
Handford
,
Michael A.
Mcdonough
,
Christopher J.
Schofield
Open Access
Abstract: AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1–3, 2–4, 5–6 disulfide bonding pattern; an unexpected Cys3–4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG oxygenases.
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Oct 2019
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I04-Macromolecular Crystallography
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Open Access
Abstract: The human 2-oxoglutarate dependent oxygenase aspartate/asparagine-β-hydroxylase (AspH) catalyses the hydroxylation of Asp/Asn-residues in epidermal growth factor-like domains (EGFDs). AspH is upregulated on the surface of malign cancer cells; increased AspH levels correlate with tumour invasiveness. Due to a lack of efficient assays to monitor the activity of isolated AspH, there are few reports of studies aimed at identifying small-molecule AspH inhibitors. Recently, it was reported that AspH substrates have a non-canonical EGFD disulfide pattern. Here we report that a stable synthetic thioether mimic of AspH substrates can be employed in solid phase extraction mass spectrometry based high-throughput AspH inhibition assays which are of excellent robustness, as indicated by high Z’-factors and good signal-to-noise/background ratios. The AspH inhibition assay was applied to screen approximately 1500 bioactive small-molecules, including natural products and active pharmaceutical ingredients of approved human therapeutics. Potent AspH inhibitors were identified from both compound classes. Our AspH inhibition assay should enable the development of potent and selective small-molecule AspH inhibitors and contribute towards the development of safer inhibitors for other 2OG oxygenases, e.g. screens of the hypoxia-inducible factor prolyl-hydroxylase inhibitors revealed that vadadustat inhibits AspH with moderate potency.
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May 2020
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