I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12346]
Open Access
Abstract: Crystallization is the bottleneck in macromolecular crystallography; even when a protein crystallises, crystal packing often influences ligand-binding and protein–protein interaction interfaces, which are the key points of interest for functional and drug discovery studies. The human hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) readily crystallises as a homotrimer, but with a sterically blocked active site. We explored strategies aimed at altering PHD2 crystal packing by protein modification and molecules that bind at its active site and elsewhere. Following the observation that, despite weak inhibition/binding in solution, succinamic acid derivatives readily enable PHD2 crystallization, we explored methods to induce crystallization without active site binding. Cyclic peptides obtained via mRNA display bind PHD2 tightly away from the active site. They efficiently enable PHD2 crystallization in different forms, both with/without substrates, apparently by promoting oligomerization involving binding to the C-terminal region. Although our work involves a specific case study, together with those of others, the results suggest that mRNA display-derived cyclic peptides may be useful in challenging protein crystallization cases.
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Dec 2020
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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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I03-Macromolecular Crystallography
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Open Access
Abstract: The 2-oxoglutarate (2OG) dependent hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) are targets for treatment of anaemia and other ischaemia related diseases. PHD inhibitors are in clinical trials; however, the number of reported templates for PHD inhibition is limited. We report structure–activity relationship and crystallographic studies on spiro[4.5]decanone containing PHD inhibitors. Together with other studies, our results reveal spiro[4.5]decanones as useful templates for generation of potent and selective 2OG oxygenase inhibitors.
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Apr 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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