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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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Vincent
Fagan
,
Catrine
Johansson
,
Carina
Gileadi
,
Octovia
Monteiro
,
James Edward
Dunford
,
Reshma
Nibhani
,
Martin
Philpott
,
Jessica
Malzahn
,
Graham
Wells
,
Ruth
Farham
,
Adam
Cribbs
,
Nadia
Halidi
,
Fengling
Li
,
Irene
Chau
,
Holger
Greschik
,
Srikannathasan
Velupillai
,
Abdellah
Allali-Hassani
,
James M.
Bennett
,
Thomas
Christott
,
Charline
Giroud
,
Andrew M.
Lewis
,
Kilian V. M.
Huber
,
Nick
Athanasou
,
Chas
Bountra
,
Manfred
Jung
,
Roland
Schüle
,
Masoud
Vedadi
,
Cheryl H.
Arrowsmith
,
Yan
Xiong
,
Jian
Jin
,
Oleg
Fedorov
,
Gillian
Farnie
,
Paul E.
Brennan
,
Udo C. T.
Oppermann
Diamond Proposal Number(s):
[10619, 15433]
Abstract: Modifications of histone tails, including lysine/arginine methylation, provide the basis of a 'chromatin or histone code'. Proteins that con-tain 'reader' domains can bind to these modifications and form specific effector complexes, which ultimately mediate chromatin function. The spindlin1 (SPIN1) protein contains three Tudor methyl-lysine/arginine reader domains and was identified as a putative onco-gene and transcriptional co-activator. Here we report a SPIN1 chemi-cal probe inhibitor with low nanomolar in vitro activity, exquisite selectivity on a panel of methyl reader and writer proteins, and with submicromolar cellular activity. X-ray crystallography showed that this Tudor domain chemical probe simultaneously engages Tudor domains 1 and 2 via a bidentate binding mode. Small molecule inhibition and siRNA knockdown of SPIN1, as well as chemoproteomic studies, iden-tified genes which are transcriptionally regulated by SPIN1 in squa-mous cell carcinoma and suggest that SPIN1 may have a roll in cancer related inflammation and/or cancer metastasis.
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Sep 2019
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I04-Macromolecular Crystallography
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Yan
Xiong
,
Holger
Greschik
,
Catrine
Johansson
,
Ludwig
Seifert
,
Johannes
Bacher
,
Kwang-Su
Park
,
Nicolas
Babault
,
Michael L.
Martini
,
Vincent
Fagan
,
Fengling
Li
,
Irene
Chau
,
Thomas
Christott
,
David
Dilworth
,
Dalia
Barsyte-Lovejoy
,
Masoud
Vedadi
,
Cheryl H.
Arrowsmith
,
Paul E.
Brennan
,
Oleg
Fedorov
,
Manfred
Jung
,
Gillian
Farnie
,
Jing
Liu
,
Udo C. T.
Oppermann
,
Roland
Schüle
,
Jian
Jin
Abstract: By screening an epigenetic compound library, we identified that UNC0638, a highly potent inhibitor of the histone methyltransferases G9a and GLP, was a weak inhibitor of SPIN1 (Spindlin 1), a methyllysine reader protein. Our optimization of this weak hit resulted in the discovery of a potent, selective and cell-active SPIN1 inhibitor, compound 3 (MS31). Compound 3 potently inhibited binding of trimethyllysine-containing peptides to SPIN1, displayed high binding affinity, was highly selective for SPIN1 over other epigenetic readers and writers, directly engaged SPIN1 in cells, and was not toxic to non-tumorigenic cells. The crystal structure of the SPIN1–compound 3 complex indicated that it selectively binds Tudor domain II of SPIN1. We also designed a structurally similar but inactive compound 4 (MS31N) as a negative control. Our results have demonstrated for the first time that potent, selective and cell-active fragment-like inhibitors can be generated by targeting a single Tudor domain.
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Jul 2019
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[1221, 7864, 9948, 15433]
Open Access
Abstract: Biosynthesis of 6-deoxy sugars, including L-fucose, involves a mechanistically complex, enzymatic 4,6-dehydration of hex-ose nucleotide precursors as the first committed step. Here, we determined pre- and post-catalytic complex structures of the human GDP-mannose 4,6-dehydratase at atomic resolution. These structures together with results of molecular dynamics simulation and biochemical characterization of wildtype and mutant enzymes reveal elusive mechanistic details of water elimination from GDP-mannose C5’’ and C6’’, coupled to NADP-mediated hydride transfer from C4’’ to C6’’. We show that concerted acid-base catalysis from only two active-site groups, Tyr179 and Glu157, promotes a syn 1,4-elimination from an enol (not an enolate) intermediate. We also show that the overall multistep catalytic reaction involves least position changes of enzyme and substrate groups; and that it proceeds under conserved exploitation of the basic (minimal) catalytic machinery of short-chain dehydrogenase/reductases.
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Mar 2019
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Joao R. C.
Muniz
,
Natalie Wing-Sum
Szeto
,
Rebecca
Frise
,
Wen Hwa
Lee
,
Xian-Song
Wang
,
Beat
Thöny
,
Nastassja
Himmelreich
,
Nenad
Blau
,
Kwang-Jen
Hsiao
,
Tze-Tze
Liu
,
Opher
Gileadi
,
Udo
Oppermann
,
Frank
Von Delft
,
Wyatt
Yue
,
Nelson Leung-Sang
Tang
Abstract: Genetic defects on 6-pyruvoyl-tetrahydropterin synthase (PTPS) are the most prevalent cause of hyperphenylalaninaemia not due to phenylalanine hydrolyase deficiency (phenylketonuria). PTPS catalyses the second step of tetrahydrobiopterin (BH4) cofactor biosynthesis, and its deficiency represents the most common form of BH4 deficiency. Untreated PTPS deficiency results in depletion of the neurotransmitters dopamine, catecholamine and serotonin causing neurological symptoms.
We archived reported missense variants of the PTS gene. Common in silico algorithms were used to predict the effects of such variants, and substantial proportions (up to 19%) of the variants were falsely classified as benign or uncertain. We have determined the crystal structure of the human PTPS hexamer, allowing another level of interpretation to understand the potential deleterious consequences of the variants from a structural perspective. The in silico and structure approaches appear to be complimentary and may provide new insights that are not available from each alone. Information from the protein structure suggested that the variants affecting amino acid residues required for interaction between monomeric subunits of the PTPS hexamer were those misclassified as benign by in silico algorithms. Our findings illustrate the important utility of 3D protein structure in interpretation of variants and also current limitations of in silico prediction algorithms. However, software to analyse mutation in the perspective of 3D protein structure is far less readily available than other in silico prediction tools.
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Mar 2019
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I04-Macromolecular Crystallography
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Saleta
Vazquez-Rodriguez
,
Miranda
Wright
,
Catherine M.
Rogers
,
Adam P.
Cribbs
,
Srikannathasan
Velupillai
,
Martin
Philpott
,
Henry
Lee
,
James E.
Dunford
,
Kilian V. M.
Huber
,
Matthew B.
Robers
,
James D.
Vasta
,
Marie-Laetitia
Thezenas
,
Sarah
Bonham
,
Benedikt
Kessler
,
James
Bennett
,
Oleg
Fedorov
,
Florence
Raynaud
,
Adam
Donovan
,
Julian
Blagg
,
Vassilios
Bavetsias
,
Udo
Oppermann
,
Chas
Bountra
,
Akane
Kawamura
,
Paul E.
Brennan
Diamond Proposal Number(s):
[15433]
Open Access
Abstract: Histone lysine demethylases (KDMs) are involved in the dynamic regulation of gene expression and they play a critical role in several biological processes. Achieving selectivity over the different KDMs has been a major challenge for KDM inhibitor development. Here we report potent and selective KDM5 covalent inhibitors designed to target cysteine residues only present in the KDM5 sub‐family. The covalent binding to the targeted proteins was confirmed by MS and time‐dependent inhibition. Additional competition assays show that compounds were non 2‐OG competitive. Target engagement and ChIP‐seq analysis showed that the compounds inhibited the KDM5 members in cells at nano‐ to micromolar levels and induce a global increase of the H3K4me3 mark at transcriptional start sites.
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Jan 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Stephanie
Oerum
,
Martine
Roovers
,
Robert
Rambo
,
Jola
Kopec
,
Henry
Bailey
,
Fiona
Fitzpatrick
,
Joseph A.
Newman
,
William G.
Newman
,
Albert
Amberger
,
Johannes
Zschocke
,
Louis
Droogmans
,
Udo
Oppermann
,
Wyatt W.
Yue
Open Access
Abstract: Mitochondrial tRNAs are transcribed as long polycistronic transcripts of precursor tRNAs and undergo posttranscriptional modifications such as endonucleolytic processing and methylation required for their correct structure and function. Among them, 5'-end processing and purine 9 N1-methylation of mitochondrial tRNA are catalysed by two proteinaceous complexes with overlapping subunit composition. The Mg2+-dependent ribonuclease P complex for 5'-end cleavage comprises the methyltransferase domain-containing protein TRMT10C/MRPP1, short-chain oxidoreductase HSD17B10/MRPP2, and metallonuclease KIAA0391/MRPP3. An MRPP1-MRPP2 sub-complex also catalyses the formation of 1-methyladenosine/1-methylguanosine at position 9 using S-adenosyl-L-methionine as methyl donor. However, a lack of structural information has precluded insights into how these complexes methylate and process mitochondrial tRNA. Here, we used a combination of X-ray crystallography, interaction and activity assays, and small angle X-ray scattering (SAXS), to gain structural insight into the two tRNA modification complexes and their components. The MRPP1 N-terminus is involved in tRNA binding and monomer-monomer self-interaction, while the C-terminal SPOUT fold contains key residues for S-adenosyl-L-methionine binding and N1-methylation. The entirety of MRPP1 interacts with MRPP2 to form the N1-methylation complex, while the MRPP1-MRPP2-MRPP3 ribonuclease P complex only assembles in the presence of precursor tRNA. This study proposes low-resolution models of the MRPP1-MRPP2 and MRPP1-MRPP2-MRPP3 complexes that suggest the overall architecture, stoichiometry, and orientation of subunits and tRNA substrates.
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Jun 2018
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Hanna
Tarhonskaya
,
Radoslaw P.
Nowak
,
Catrine
Johansson
,
Aleksandra
Szykowska
,
Anthony
Tumber
,
Rebecca L.
Hancock
,
Pauline
Lang
,
Emily
Flashman
,
Udo
Oppermann
,
Christopher J.
Schofield
,
Akane
Kawamura
Diamond Proposal Number(s):
[11175, 10619]
Open Access
Abstract: Methylation of lysine-4 of histone H3 (H3K4men) is an important regulatory factor in eukaryotic transcription. Removal of the transcriptionally activating H3K4 methylation is catalysed by histone demethylases, including the JmjC KDM5 subfamily. The JmjC KDMs are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases, some of which are associated with cancer. Altered levels of TCA cycle intermediates, and the associated metabolites D- and L-2-hydroxyglutarate (2HG), can cause changes in chromatin methylation status. We report comprehensive biochemical, structural and cellular studies on the interaction of TCA cycle intermediates with KDM5B which is a current medicinal chemistry target for cancer. The tested TCA intermediates were poor or moderate KDM5B inhibitors, except for oxaloacetate and succinate, which were shown to compete for binding with 2OG. D- and L-2HG were moderately potent inhibitors at levels which might be relevant in cancer cells bearing isocitrate dehydrogenase mutations. Crystallographic analyses with succinate, fumarate, L-malate, oxaloacetate, pyruvate, D- and L-2HG support the kinetic studies showing competition with 2OG. An unexpected binding mode for oxaloacetate was observed in which it coordinates the active site metal via its C-4 carboxylate rather than the C-1 carboxylate/C-2 keto groups. Studies employing immunofluorescence antibody-based assays reveal no changes in H3K4me3 levels in cells ectopically overexpressing KDM5B in response to dosing with TCA cycle metabolite pro-drug esters, suggesting that the high levels of cellular 2OG may preclude inhibition. The combined results reveal the potential for KDM5B inhibition by TCA cycle intermediates, but suggest that in cells such inhibition will normally be effectively competed by 2OG.
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Aug 2017
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I02-Macromolecular Crystallography
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Anthony
Tumber
,
Andrea
Nuzzi
,
Edward S.
Hookway
,
Stephanie B.
Hatch
,
Srikannathasan
Velupillai
,
Catrine
Johansson
,
Akane
Kawamura
,
Pavel
Savitsky
,
Clarence
Yapp
,
Aleksandra
Szykowska
,
Na
Wu
,
Chas
Bountra
,
Claire
Strain-Damerell
,
Nicola A.
Burgess-Brown
,
Gian Filippo
Ruda
,
Oleg
Fedorov
,
Shonagh
Munro
,
Katherine S.
England
,
Radoslaw P.
Nowak
,
Christopher J.
Schofield
,
Nicholas B.
La Thangue
,
Charlotte
Pawlyn
,
Faith
Davies
,
Gareth
Morgan
,
Nick
Athanasou
,
Susanne
Müller
,
Udo
Oppermann
,
Paul E.
Brennan
Open Access
Abstract: Methylation of lysine residues on histone tail is a dynamic epigenetic modification that plays a key role in chromatin structure and gene regulation. Members of the KDM5 (also known as JARID1) sub-family are 2-oxoglutarate (2-OG) and Fe2+-dependent oxygenases acting as histone 3 lysine 4 trimethyl (H3K4me3) demethylases, regulating proliferation, stem cell self-renewal, and differentiation. Here we present the characterization of KDOAM-25, an inhibitor of KDM5 enzymes. KDOAM-25 shows biochemical half maximal inhibitory concentration values of <100 nM for KDM5A-D in vitro, high selectivity toward other 2-OG oxygenases sub-families, and no off-target activity on a panel of 55 receptors and enzymes. In human cell assay systems, KDOAM-25 has a half maximal effective concentration of ∼50 μM and good selectivity toward other demethylases. KDM5B is overexpressed in multiple myeloma and negatively correlated with the overall survival. Multiple myeloma MM1S cells treated with KDOAM-25 show increased global H3K4 methylation at transcriptional start sites and impaired proliferation.
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Mar 2017
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B21-High Throughput SAXS
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Catrine
Johansson
,
Velupillai
Srikannathasan
,
Anthony
Tumber
,
Aleksandra
Szykowska
,
Edward S.
Hookway
,
Radoslaw
Nowak
,
Claire
Strain-Damerell
,
Carina
Gileadi
,
Martin
Philpott
,
Nicola
Burgess-Brown
,
Na
Wu
,
Jolanta
Kopec
,
Andrea
Nuzzi
,
Holger
Steuber
,
Ursula
Egner
,
Volker
Badock
,
Shonagh
Munro
,
Nicholas B
Lathangue
,
Sue
Westaway
,
Jack
Brown
,
Nick
Athanasou
,
Rab
Prinjha
,
Paul E
Brennan
,
Udo
Oppermann
Diamond Proposal Number(s):
[10619]
Abstract: Members of the KDM5 (also known as JARID1) family are 2-oxoglutarate- and Fe2+-dependent oxygenases that act as histone H3K4 demethylases, thereby regulating cell proliferation and stem cell self-renewal and differentiation. Here we report crystal structures of the catalytic core of the human KDM5B enzyme in complex with three inhibitor chemotypes. These scaffolds exploit several aspects of the KDM5 active site, and their selectivity profiles reflect their hybrid features with respect to the KDM4 and KDM6 families. Whereas GSK-J1, a previously identified KDM6 inhibitor, showed about sevenfold less inhibitory activity toward KDM5B than toward KDM6 proteins, KDM5-C49 displayed 25–100-fold selectivity between KDM5B and KDM6B. The cell-permeable derivative KDM5-C70 had an antiproliferative effect in myeloma cells, leading to genome-wide elevation of H3K4me3 levels. The selective inhibitor GSK467 exploited unique binding modes, but it lacked cellular potency in the myeloma system. Taken together, these structural leads deliver multiple starting points for further rational and selective inhibitor design.
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May 2016
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