I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Giancarlo
Abis
,
Rebecca L.
Charles
,
Jolanta
Kopec
,
Wyatt W.
Yue
,
R. Andrew
Atkinson
,
Tam T. T.
Bui
,
Steven
Lynham
,
Simona
Popova
,
Yin-Biao
Sun
,
Franca
Fraternali
,
Philip
Eaton
,
Maria R.
Conte
Diamond Proposal Number(s):
[13597, 10619]
Open Access
Abstract: Human soluble epoxide hydrolase (hsEH) is an enzyme responsible for the inactivation of bioactive epoxy fatty acids, and its inhibition is emerging as a promising therapeutical strategy to target hypertension, cardiovascular disease, pain and insulin sensitivity. Here, we uncover the molecular bases of hsEH inhibition mediated by the endogenous 15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2). Our data reveal a dual inhibitory mechanism, whereby hsEH can be inhibited by reversible docking of 15d-PGJ2 in the catalytic pocket, as well as by covalent locking of the same compound onto cysteine residues C423 and C522, remote to the active site. Biophysical characterisations allied with in silico investigations indicate that the covalent modification of the reactive cysteines may be part of a hitherto undiscovered allosteric regulatory mechanism of the enzyme. This study provides insights into the molecular modes of inhibition of hsEH epoxy-hydrolytic activity and paves the way for the development of new allosteric inhibitors.
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May 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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B21-High Throughput SAXS
I04-1-Macromolecular Crystallography (fixed wavelength)
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D. Sean
Froese
,
Jolanta
Kopec
,
Elzbieta
Rembeza
,
Gustavo
Arruda Bezerra
,
Anselm Erich
Oberholzer
,
Terttu
Suormala
,
Seraina
Lutz
,
Rod
Chalk
,
Oktawia
Borkowska
,
Matthias R.
Baumgartner
,
Wyatt W.
Yue
Diamond Proposal Number(s):
[10619, 51433]
Open Access
Abstract: The folate and methionine cycles are crucial for biosynthesis of lipids, nucleotides and proteins, and production of the methyl donor S-adenosylmethionine (SAM). 5,10-methylenetetrahydrofolate reductase (MTHFR) represents a key regulatory connection between these cycles, generating 5-methyltetrahydrofolate for initiation of the methionine cycle, and undergoing allosteric inhibition by its end product SAM. Our 2.5 Å resolution crystal structure of human MTHFR reveals a unique architecture, appending the well-conserved catalytic TIM-barrel to a eukaryote-only SAM-binding domain. The latter domain of novel fold provides the predominant interface for MTHFR homo-dimerization, positioning the N-terminal serine-rich phosphorylation region near the C-terminal SAM-binding domain. This explains how MTHFR phosphorylation, identified on 11 N-terminal residues (16 in total), increases sensitivity to SAM binding and inhibition. Finally, we demonstrate that the 25-amino-acid inter-domain linker enables conformational plasticity and propose it to be a key mediator of SAM regulation. Together, these results provide insight into the molecular regulation of MTHFR.
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Jun 2018
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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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B21-High Throughput SAXS
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10619]
Open Access
Abstract: The multi-domain enzyme phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of dietary I-phenylalanine (Phe) to I-tyrosine. Inherited mutations that result in PAH enzyme deficiency are the genetic cause of the autosomal recessive disorder phenylketonuria. Phe is the substrate for the PAH active site, but also an allosteric ligand that increases enzyme activity. Phe has been proposed to bind, in addition to the catalytic domain, a site at the PAH N-terminal regulatory domain (PAH-RD), to activate the enzyme via an unclear mechanism. Here we report the crystal structure of human PAH-RD bound with Phe at 1.8 Å resolution, revealing a homodimer of ACT folds with Phe bound at the dimer interface. This work delivers the structural evidence to support previous solution studies that a binding site exists in the RD for Phe, and that Phe binding results in dimerization of PAH-RD. Consistent with our structural observation, a disease-associated PAH mutant impaired in Phe binding disrupts the monomer:dimer equilibrium of PAH-RD. Our data therefore support an emerging model of PAH allosteric regulation, whereby Phe binds to PAH-RD and mediates the dimerization of regulatory modules that would bring about conformational changes to activate the enzyme.
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Apr 2016
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10619]
Open Access
Abstract: Classic galactosemia is a potentially lethal disease caused by the dysfunction of galactose 1-phosphate uridylyltransferase (GALT). Over 300 disease-associated GALT mutations have been reported, with the majority being missense changes, although a better understanding of their underlying molecular effects has been hindered by the lack of structural information for the human enzyme. Here, we present the 1.9 Å resolution crystal structure of human GALT (hGALT) ternary complex, revealing a homodimer arrangement that contains a covalent uridylylated intermediate and glucose-1-phosphate in the active site, as well as a structural zinc-binding site, per monomer. hGALT reveals significant structural differences from bacterial GALT homologues in metal ligation and dimer interactions, and therefore is a zbetter model for understanding the molecular consequences of disease mutations. Both uridylylation and zinc binding influence the stability and aggregation tendency of hGALT. This has implications for disease-associated variants where p.Gln188Arg, the most commonly detected, increases the rate of aggregation in the absence of zinc likely due to its reduced ability to form the uridylylated intermediate. As such our structure serves as a template in the future design of pharmacological chaperone therapies and opens new concepts about the roles of metal binding and activity in protein misfolding by disease-associated mutants.
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Mar 2016
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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C. X.
Santos
,
A. D.
Hafstad
,
M.
Beretta
,
M.
Zhang
,
C.
Molenaar
,
J.
Kopec
,
D.
Fotinou
,
T. V.
Murray
,
A. M.
Cobb
,
D.
Martin
,
M.
Zeh Silva
,
N.
Anilkumar
,
K.
Schroder
,
C. M.
Shanahan
,
A. C.
Brewer
,
R. P.
Brandes
,
E.
Blanc
,
M.
Parsons
,
V.
Belousov
,
R.
Cammack
,
R. C.
Hider
,
R. A.
Steiner
,
A. M.
Shah
Diamond Proposal Number(s):
[7656]
Open Access
Abstract: Phosphorylation of translation initiation factor 2α (eIF2α) attenuates global protein synthesis but enhances translation of activating transcription factor 4 (ATF4) and is a crucial evolutionarily conserved adaptive pathway during cellular stresses. The serine–threonine protein phosphatase 1 (PP1) deactivates this pathway whereas prolonging eIF2α phosphorylation enhances cell survival. Here, we show that the reactive oxygen species‐generating NADPH oxidase‐4 (Nox4) is induced downstream of ATF4, binds to a PP1‐targeting subunit GADD34 at the endoplasmic reticulum, and inhibits PP1 activity to increase eIF2α phosphorylation and ATF4 levels. Other PP1 targets distant from the endoplasmic reticulum are unaffected, indicating a spatially confined inhibition of the phosphatase. PP1 inhibition involves metal center oxidation rather than the thiol oxidation that underlies redox inhibition of protein tyrosine phosphatases. We show that this Nox4‐regulated pathway robustly enhances cell survival and has a physiologic role in heart ischemia–reperfusion and acute kidney injury. This work uncovers a novel redox signaling pathway, involving Nox4–GADD34 interaction and a targeted oxidative inactivation of the PP1 metal center, that sustains eIF2α phosphorylation to protect tissues under stress.
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Jan 2016
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I03-Macromolecular Crystallography
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Moniek
Riemersma
,
Sean
Froese
,
Walinka
Van tol
,
Udo f
Engelke
,
Jolanta
Kopec
,
Monique
Van scherpenzeel
,
Angel
Ashikov
,
Tobias
Krojer
,
Frank
Von Delft
,
Marco
Tessari
,
Anna
Buczkowska
,
Ewa
Swiezewska
,
Lucas t
Jae
,
Thijn r
Brummelkamp
,
Hiroshi
Manya
,
Tamao
Endo
,
Hans
Van bokhoven
,
Wyatt
Yue
,
Dirk j
Lefeber
Abstract: A unique, unsolved O-mannosyl glycan on alpha-dystroglycan is essential for its interaction with protein ligands in the extracellular matrix. Defective O-mannosylation leads to a group of muscular dystrophies, called dystroglycanopathies. Mutations in isoprenoid synthase domain containing (ISPD) represent the second most common cause of these disorders, however, its molecular function remains uncharacterized. The human ISPD (hISPD) crystal structure showed a canonical N-terminal cytidyltransferase domain linked to a C-terminal domain that is absent in cytidyltransferase homologs. Functional studies demonstrated cytosolic localization of hISPD, and cytidyltransferase activity toward pentose phosphates, including ribulose 5-phosphate, ribose 5-phosphate, and ribitol 5-phosphate. Identity of the CDP sugars was confirmed by liquid chromatography quadrupole time-of-flight mass spectrometry and two-dimensional nuclear magnetic resonance spectroscopy. Our combined results indicate that hISPD is a cytidyltransferase, suggesting the presence of a novel human nucleotide sugar essential for functional alpha-dystroglycan O-mannosylation in muscle and brain. Thereby, ISPD deficiency can be added to the growing list of tertiary dystroglycanopathies.
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Dec 2015
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B21-High Throughput SAXS
I04-Macromolecular Crystallography
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Sean
Froese
,
Jolanta
Kopec
,
Fiona
Fitzpatrick
,
Marion
Schuller
,
Thomas
Mccorvie
,
Rod
Chalk
,
Tanja
Plessl
,
Victoria
Fettelschoss
,
Brian
Fowler
,
Matthias R.
Baumgartner
,
Wyatt
Yue
Diamond Proposal Number(s):
[10619]
Open Access
Abstract: Background: Two intracellular proteins,
MMACHC and MMADHC, functionally interact
for cobalamin trafficking.
Results: MMADHC crystal structure reveals
protein-interacting regions and unexpected
homology to MMACHC; mutations on either
protein interfere with complex formation via
different mechanisms.
Conclusion: Complex formation likely depends on
prior cobalamin processing and can be broken by
disease mutations.
Significance: MMACHC-MMADHC
heterodimerization forms the essential trafficking
chaperone delivering cobalamin to client enzymes.
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Oct 2015
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8421]
Open Access
Abstract: Periodontitis (PD) is a known risk factor for rheumatoid arthritis (RA) and there is increasing evidence that the link between the two diseases is due to citrullination by the unique bacterial peptidylarginine deiminase (PAD) enzyme expressed by periodontal pathogen Pophyromonas gingivalis (PPAD). However, the precise mechanism by which PPAD could generate potentially immunogenic peptides has remained controversial due to lack of information about the structural and catalytic mechanisms of the enzyme.
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Jul 2015
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