I03-Macromolecular Crystallography
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Tongri
Liu
,
Martine I.
Abboud
,
Rasheduzzaman
Chowdhury
,
Anthony
Tumber
,
Adam P.
Hardy
,
Kerstin
Lippl
,
Christopher T.
Lohans
,
Elisabete
Pires
,
James
Wickens
,
Michael
Mcdonough
,
Christopher M.
West
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[12346]
Abstract: In animals, the response to chronic hypoxia is mediated by prolyl-hydroxylases (PHDs) that regulate the levels of hypoxia inducible transcription factor a (HIFα). PHD homologues exist in other types of eukaryotes and prokaryotes where they act on non-HIF substrates. To gain insight into the factors underlying different PHD substrates and properties, we carried out biochemical and biophysical studies on PHD homologues from the slime mold, Dictyostelium discoideum, and the protozoan parasite, Toxoplasma gondii, both lacking HIF. The respective prolyl-hydroxylases (DdPhyA and TgPhyA) catalyze prolyl-hydroxylation of S-Phase Kinase Associated Protein 1 (Skp1), a reaction enabling adaptation to different dioxygen availability. Assays with full length Skp1 substrates reveal substantial differences in the kinetic properties of DdPhyA and TgPhyA, both with respect to each other and compared with human PHD2; consistent with cellular studies TgPhyA is more active at low dioxygen concentrations than DdPhyA. TgSkp1 is a DdPhyA substrate and DdSkp1 is a TgPhyA substrate. No cross-reactivity was detected between DdPhyA/TgPhyA substrates and human PHD2. The human Skp1 E147P variant is a DdPhyA and TgPhyA substrate, suggesting some retention of ancestral interactions. Crystallographic analysis of DdPhyA enables comparisons with homologues from humans, Trichoplax adhaerens, and prokaryotes, TgPhyA informing on differences in mobile elements involved in substrate binding and catalysis. In DdPhyA, two mobile loops that enclose substrates in the PHDs are conserved, but the C-terminal helix of the PHDs is strikingly absent. The combined results support the proposal that PHD homologues have evolved kinetic and structural features suited to their specific sensing roles.
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Sep 2020
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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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I03-Macromolecular Crystallography
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James P.
Holt‐martyn
,
Rasheduzzaman
Chowdhury
,
Anthony
Tumber
,
Tzu‐lan
Yeh
,
Martine I.
Abboud
,
Kerstin
Lippl
,
Christopher T.
Lohans
,
Gareth W.
Langley
,
William
Figg
,
Michael A.
Mcdonough
,
Christopher W.
Pugh
,
Peter J.
Ratcliffe
,
Christopher J.
Schofield
Abstract: The 2‐oxoglutarate‐dependent hypoxia inducible factor prolyl hydroxylases (PHDs) are targets for treatment of a variety of diseases including anaemia. One PHD inhibitor is approved for use for the treatment of renal anaemia and others are in late stage clinical trials. The number of reported templates for PHD inhibition is limited. We report structure–activity relationship and crystallographic studies on a promising class of 4‐hydroxypyrimidine‐containing PHD inhibitors.
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Dec 2019
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12346]
Abstract: JmjC domain containing protein 6 (JMJD6) is a 2-oxoglutarate (2OG)-dependent oxygenase linked to various cellular processes including splicing regulation, histone modification, transcriptional pause release, hypoxia sensing, and cancer. JMJD6 is reported to catalyze hydroxylation of lysine residue(s) of histones, the tumor suppressor protein p53, and splicing regulatory proteins, including u2 small nuclear ribonucleoprotein auxiliary factor 65-kDa subunit (U2AF65). JMJD6 is also reported to catalyze N-demethylation of N-methylated (both mono- and di-methylated) arginine residues of histones, and other proteins including heat shock protein 70 (HSP70), oestrogen receptor α (ERα) and RNA helicase A. Here we report MS- and NMR-based kinetic assays employing purified JMJD6 and multiple substrate fragment sequences, the results of which support the assignment of purified JMJD6 as a lysyl hydroxylase. By contrast, we did not observe N-methyl arginyl N-demethylation with purified JMJD6. Biophysical analyses including crystallographic analyses of JMJD6Δ344-403 in complex with iron and 2OG supported its assignment as a lysyl-hydroxylase rather than an N-methyl arginyl-demethylase. The screening results supported some, but not all, of the assigned JMJD6 substrates and identified other potential JMJD6 substrates. We envision these results will be useful in cellular and biological work on the substrates and functions of JMJD6 and in the development of selective inhibitors of human 2OG oxygenases.
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May 2019
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[1230]
Open Access
Abstract: Background: In humans and other animals, the chronic hypoxic response is mediated by hypoxia inducible transcription factors (HIFs) which regulate the expression of genes that counteract the effects of limiting oxygen. Prolyl hydroxylases (PHDs) act as hypoxia sensors for the HIF system in organisms ranging from humans to the simplest animal Trichoplax adhaerens.
Methods: We report structural and biochemical studies on the T. adhaerens HIF prolyl hydroxylase (TaPHD) that inform about the evolution of hypoxia sensing in animals.
Results: High resolution crystal structures (≤1.3 Å) of TaPHD, with and without its HIFα substrate, reveal remarkable conservation of key active site elements between T. adhaerens and human PHDs, which also manifest in kinetic comparisons.
Conclusion: Conserved structural features of TaPHD and human PHDs include those apparently enabling the slow binding/reaction of oxygen with the active site Fe(II), the formation of a stable 2-oxoglutarate complex, and a stereoelectronically promoted change in conformation of the hydroxylated proline-residue. Comparison of substrate selectivity between the human PHDs and TaPHD provides insights into the selectivity determinants of HIF binding by the PHDs, and into the evolution of the multiple HIFs and PHDs present in higher animals.
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Nov 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[18069]
Open Access
Abstract: N‐Methylation of lysyl residues is widely observed on histone proteins. Using isolated enzymes, we report mechanistic and structural studies on histone lysine demethylase (KDM)‐catalysed demethylation of Nε‐methylated lysine 26 on histone 1 isotype 4 (H1.4). The results reveal that methylated H1.4K26 is a substrate for all members of the KDM4 subfamily and that KDM4A‐catalysed demethylation of H1.4K26me3 peptide is similarly efficient to that of H3K9me3. Crystallographic studies of an H1.4K26me3:KDM4A complex reveal a conserved binding geometry to that of H3K9me3. In the light of the high activity of the KDM4s on this mark, our results suggest JmjC KDM‐catalysed demethylation of H1.4K26 may be as prevalent as demethylation on the H3 tail and warrants further investigation in cells.
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Oct 2018
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I04-Macromolecular Crystallography
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Suzana
Markolovic
,
Qinqin
Zhuang
,
Sarah E.
Wilkins
,
Charlotte D.
Eaton
,
Martine I.
Abboud
,
Maximiliano J.
Katz
,
Helen E.
Mcneil
,
Robert K.
Leśniak
,
Charlotte
Hall
,
Weston B.
Struwe
,
Rebecca
Konietzny
,
Simon
Davis
,
Ming
Yang
,
Wei
Ge
,
Justin L. P.
Benesch
,
Benedikt M.
Kessler
,
Peter J.
Ratcliffe
,
Matthew E.
Cockman
,
Roman
Fischer
,
Pablo
Wappner
,
Rasheduzzaman
Chowdhury
,
Mathew L.
Coleman
,
Christopher J.
Schofield
Abstract: Biochemical, structural and cellular studies reveal Jumonji-C (JmjC) domain-containing 7 (JMJD7) to be a 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes (3S)-lysyl hydroxylation. Crystallographic analyses reveal JMJD7 to be more closely related to the JmjC hydroxylases than to the JmjC demethylases. Biophysical and mutation studies show that JMJD7 has a unique dimerization mode, with interactions between monomers involving both N- and C-terminal regions and disulfide bond formation. A proteomic approach identifies two related members of the translation factor (TRAFAC) family of GTPases, developmentally regulated GTP-binding proteins 1 and 2 (DRG1/2), as activity-dependent JMJD7 interactors. Mass spectrometric analyses demonstrate that JMJD7 catalyzes Fe(ii)- and 2OG-dependent hydroxylation of a highly conserved lysine residue in DRG1/2; amino-acid analyses reveal that JMJD7 catalyzes (3S)-lysyl hydroxylation. The functional assignment of JMJD7 will enable future studies to define the role of DRG hydroxylation in cell growth and disease.
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Jun 2018
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I03-Macromolecular Crystallography
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Open Access
Abstract: Oxygenase-catalysed post-translational modifications of basic protein residues, including lysyl hydroxylations and Nε-methyl lysyl demethylations, have important cellular roles. Jumonji-C (JmjC) domain-containing protein 5 (JMJD5), which genetic studies reveal is essential in animal development, is reported as a histone Nε-methyl lysine demethylase (KDM). Here we report how extensive screening with peptides based on JMJD5 interacting proteins led to the finding that JMJD5 catalyses stereoselective C-3 hydroxylation of arginine residues in sequences from human regulator of chromosome condensation domain-containing protein 1 (RCCD1) and ribosomal protein S6 (RPS6). High-resolution crystallographic analyses reveal overall fold, active site and substrate binding/product release features supporting the assignment of JMJD5 as an arginine hydroxylase rather than a KDM. The results will be useful in the development of selective oxygenase inhibitors for the treatment of cancer and genetic diseases.
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Mar 2018
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Tzu-lan
Yeh
,
Thomas m.
Leissing
,
Martine I.
Abboud
,
Cyrille C.
Thinnes
,
Onur
Atasoylu
,
James P.
Holt-martyn
,
Dong
Zhang
,
Anthony
Tumber
,
Kerstin
Lippl
,
Christopher T.
Lohans
,
Ivanhoe K. H.
Leung
,
Helen
Morcrette
,
Ian J.
Clifton
,
Timothy D. W.
Claridge
,
Akane
Kawamura
,
Emily
Flashman
,
Xin
Lu
,
Peter J.
Ratcliffe
,
Rasheduzzaman
Chowdhury
,
Christopher W.
Pugh
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[12346, 9306]
Open Access
Abstract: Inhibition of the human 2-oxoglutarate (2OG) dependent hypoxia inducible factor (HIF) prolyl hydroxylases (human PHD1–3) causes upregulation of HIF, thus promoting erythropoiesis and is therefore of therapeutic interest. We describe cellular, biophysical, and biochemical studies comparing four PHD inhibitors currently in clinical trials for anaemia treatment, that describe their mechanisms of action, potency against isolated enzymes and in cells, and selectivities versus representatives of other human 2OG oxygenase subfamilies. The ‘clinical’ PHD inhibitors are potent inhibitors of PHD catalyzed hydroxylation of the HIF-α oxygen dependent degradation domains (ODDs), and selective against most, but not all, representatives of other human 2OG dependent dioxygenase subfamilies. Crystallographic and NMR studies provide insights into the different active site binding modes of the inhibitors. Cell-based results reveal the inhibitors have similar effects on the upregulation of HIF target genes, but differ in the kinetics of their effects and in extent of inhibition of hydroxylation of the N- and C-terminal ODDs; the latter differences correlate with the biophysical observations.
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Sep 2017
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Akane
Kawamura
,
Martin
Münzel
,
Tatsuya
Kojima
,
Clarence
Yapp
,
Bhaskar
Bhushan
,
Yuki
Goto
,
Anthony
Tumber
,
Takayuki
Katoh
,
Oliver N. F.
King
,
Toby
Passioura
,
Louise J.
Walport
,
Stephanie B.
Hatch
,
Sarah
Madden
,
Susanne
Müller
,
Paul E.
Brennan
,
Rasheduzzaman
Chowdhury
,
Richard J.
Hopkinson
,
Hiroaki
Suga
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[1230, 9306]
Open Access
Abstract: The JmjC histone demethylases (KDMs) are linked to tumour cell proliferation and are current cancer targets; however, very few highly selective inhibitors for these are available. Here we report cyclic peptide inhibitors of the KDM4A-C with selectivity over other KDMs/2OG oxygenases, including closely related KDM4D/E isoforms. Crystal structures and biochemical analyses of one of the inhibitors (CP2) with KDM4A reveals that CP2 binds differently to, but competes with, histone substrates in the active site. Substitution of the active site binding arginine of CP2 to N-ɛ-trimethyl-lysine or methylated arginine results in cyclic peptide substrates, indicating that KDM4s may act on non-histone substrates. Targeted modifications to CP2 based on crystallographic and mass spectrometry analyses results in variants with greater proteolytic robustness. Peptide dosing in cells manifests KDM4A target stabilization. Although further development is required to optimize cellular activity, the results reveal the feasibility of highly selective non-metal chelating, substrate-competitive inhibitors of the JmjC KDMs.
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Apr 2017
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