I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18548, 25402]
Open Access
Abstract: The liver isoform of pyruvate kinase (PKL) has gained interest due to its potential capacity to regulate fatty acid synthesis involved in the progression of non-alcoholic fatty liver disease (NAFLD). Here we describe a novel series of PKL modulators that can either activate or inhibit the enzyme allosterically, from a cryptic site at the interface of two protomers in the tetrameric enzyme. Starting from urolithin D, we designed and synthesised 42 new compounds. The effect of these compounds on PKL enzymatic activity was assessed after incubation with cell lysates obtained from a liver cell line. Pronounced activation of PKL activity, up to 3.8-fold, was observed for several compounds at 10 μM, while other compounds were prominent PKL inhibitors reducing its activity to 81% at best. A structure-activity relationship identified linear-shaped sulfone-sulfonamides as activators and non-linear compounds as inhibitors. Crystal structures revealed the conformations of these modulators, which were used as a reference for designing new modulators.
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Mar 2023
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I02-Macromolecular Crystallography
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Christos
Stergiou
,
Rhys
Williams
,
Jennifer R.
Fleming
,
Vasiliki
Zouvelou
,
Elpinickie
Ninou
,
Francesca
Andreetta
,
Elena
Rinaldi
,
Ornella
Simoncini
,
Renato
Mantegazza
,
Julius
Bogomolovas
,
John
Tzartos
,
Siegfried
Labeit
,
Olga
Mayans
,
Socrates
Tzartos
Diamond Proposal Number(s):
[8997]
Open Access
Abstract: Myasthenia gravis (MG) is an autoimmune disease caused by antibodies targeting the neuromuscular junction (NJ) of skeletal muscles. The major MG autoantigen is nicotinic acetylcholine receptor. Other autoantigens at the NJ include MuSK, LRP4 and agrin. Autoantibodies to the intra-sarcomeric striated muscle-specific gigantic protein titin, although not directed to the NJ, are invaluable biomarkers for thymoma and MG disease severity. Thymus and thymoma are critical in MG mechanisms and management. Titin autoantibodies bind to a 30 KDa titin segment, the main immunogenic region (MIR), consisting of an Ig-FnIII-FnIII 3-domain tandem, termed I109-I111. In this work, we further resolved the localization of titin epitope(s) to facilitate the development of more specific anti-titin diagnostics. For this, we expressed protein samples corresponding to 8 MIR and non-MIR titin fragments and tested 77 anti-titin sera for antibody binding using ELISA, competition experiments and Western blots. All anti-MIR antibodies were bound exclusively to the central MIR domain, I110, and to its containing titin segments. Most antibodies were bound also to SDS-denatured I110 on Western blots, suggesting that their epitope(s) are non-conformational. No significant difference was observed between thymoma and non-thymoma patients or between early- and late-onset MG. In addition, atomic 3D-structures of the MIR and its subcomponents were elucidated using X-ray crystallography. These immunological and structural data will allow further studies into the atomic determinants underlying titin-based autoimmunity, improved diagnostics and how to eventually treat titin autoimmunity associated co-morbidities.
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Feb 2023
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B21-High Throughput SAXS
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Tobias
Schmidt
,
Adrianna
Dabrowska
,
Joseph A.
Waldron
,
Kelly
Hodge
,
Grigorios
Koulouras
,
Mads
Gabrielsen
,
June
Munro
,
David C.
Tack
,
Gemma
Harris
,
Ewan
Mcghee
,
David
Scott
,
Leo m.
Carlin
,
Danny
Huang
,
John
Le quesne
,
Sara
Zanivan
,
Ania
Wilczynska
,
Martin
Bushell
Diamond Proposal Number(s):
[21657]
Open Access
Abstract: Altered eIF4A1 activity promotes translation of highly structured, eIF4A1-dependent oncogene mRNAs at root of oncogenic translational programmes. It remains unclear how these mRNAs recruit and activate eIF4A1 unwinding specifically to facilitate their preferential translation. Here, we show that single-stranded RNA sequence motifs specifically activate eIF4A1 unwinding allowing local RNA structural rearrangement and translation of eIF4A1-dependent mRNAs in cells. Our data demonstrate that eIF4A1-dependent mRNAs contain AG-rich motifs within their 5’UTR which specifically activate eIF4A1 unwinding of local RNA structure to facilitate translation. This mode of eIF4A1 regulation is used by mRNAs encoding components of mTORC-signalling and cell cycle progression, and renders these mRNAs particularly sensitive to eIF4A1-inhibition. Mechanistically, we show that binding of eIF4A1 to AG-rich sequences leads to multimerization of eIF4A1 with eIF4A1 subunits performing distinct enzymatic activities. Our structural data suggest that RNA-binding of multimeric eIF4A1 induces conformational changes in the RNA resulting in an optimal positioning of eIF4A1 proximal to the RNA duplex enabling efficient unwinding. Our data proposes a model in which AG-motifs in the 5’UTR of eIF4A1-dependent mRNAs specifically activate eIF4A1, enabling assembly of the helicase-competent multimeric eIF4A1 complex, and positioning these complexes proximal to stable localised RNA structure allowing ribosomal subunit scanning.
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Feb 2023
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Krios IV-Titan Krios IV at Diamond
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Yang
Yang
,
Holly J.
Garringer
,
Yang
Shi
,
Sofia
Lovestam
,
Sew
Peak-Chew
,
Xianjun
Zhang
,
Abhay
Kotecha
,
Mehtap
Bacioglu
,
Atsuo
Koto
,
Masaki
Takao
,
Maria Grazia
Spillantini
,
Bernardino
Ghetti
,
Ruben
Vidal
,
Alexey G.
Murzin
,
Sjors H. W.
Scheres
,
Michel
Goedert
Diamond Proposal Number(s):
[23268]
Open Access
Abstract: A 21-nucleotide duplication in one allele of SNCA was identified in a previously described disease with abundant α-synuclein inclusions that we now call juvenile-onset synucleinopathy (JOS). This mutation translates into the insertion of MAAAEKT after residue 22 of α-synuclein, resulting in a protein of 147 amino acids. Both wild-type and mutant proteins were present in sarkosyl-insoluble material that was extracted from frontal cortex of the individual with JOS and examined by electron cryo-microscopy. The structures of JOS filaments, comprising either a single protofilament, or a pair of protofilaments, revealed a new α-synuclein fold that differs from the folds of Lewy body diseases and multiple system atrophy (MSA). The JOS fold consists of a compact core, the sequence of which (residues 36–100 of wild-type α-synuclein) is unaffected by the mutation, and two disconnected density islands (A and B) of mixed sequences. There is a non-proteinaceous cofactor bound between the core and island A. The JOS fold resembles the common substructure of MSA Type I and Type II dimeric filaments, with its core segment approximating the C-terminal body of MSA protofilaments B and its islands mimicking the N-terminal arm of MSA protofilaments A. The partial similarity of JOS and MSA folds extends to the locations of their cofactor-binding sites. In vitro assembly of recombinant wild-type α-synuclein, its insertion mutant and their mixture yielded structures that were distinct from those of JOS filaments. Our findings provide insight into a possible mechanism of JOS fibrillation in which mutant α-synuclein of 147 amino acids forms a nucleus with the JOS fold, around which wild-type and mutant proteins assemble during elongation.
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Feb 2023
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I03-Macromolecular Crystallography
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Abstract: The mutation V600E in B-Raf leads to mitogen activated protein kinase (MAPK) pathway activation, uncontrolled cell proliferation, and tumorigenesis. ATP competitive type I B-Raf inhibitors, such as vemurafenib (1) and PLX4720 (4) efficiently block the MAPK pathways in B-Raf mutant cells, however these inhibitors induce conformational changes in the wild type B-Raf (wtB-Raf) kinase domain leading to heterodimerization with C-Raf, causing paradoxical hyperactivation of the MAPK pathway. This unwanted activation may be avoided by another class of inhibitors (type II) which bind the kinase in the DFG-out conformation, such as AZ628 (3) preventing heterodimerization. Here we present a new B-Raf kinase domain inhibitor, based on a phenyl(1H-pyrrolo [2,3-b]pyridin-3-yl)methanone template, that represents a hybrid between 4 and 3. This novel inhibitor borrows the hinge binding region from 4 and the back pocket binding moiety from 3. We determined its binding mode, performed activity/selectivity studies, and molecular dynamics simulations in order to study the conformational effects induced by this inhibitor on wt and V600E mutant B-Raf kinase. We discovered that the inhibitor was active and selective for B-Raf, binds in a DFG-out/αC-helix-in conformation, and did not induce the aforementioned paradoxical hyperactivation in the MAPK pathway. We propose that this merging approach can be used to design a novel class of B-Raf inhibitors for translational studies.
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Feb 2023
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I04-Macromolecular Crystallography
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James S.
Scott
,
Darren
Stead
,
Bernard
Barlaam
,
Jason
Breed
,
Rodrigo J.
Carbajo
,
Elisabetta
Chiarparin
,
Natalie
Cureton
,
Paul R. J.
Davey
,
David I.
Fisher
,
Eric T.
Gangl
,
Tyler
Grebe
,
Ryan D.
Greenwood
,
Sudhir
Hande
,
Holia
Hatoum-Mokdad
,
Samantha J.
Hughes
,
Thomas A.
Hunt
,
Tony
Johnson
,
Stefan L.
Kavanagh
,
Teresa C. M.
Klinowska
,
Carrie J. B.
Larner
,
Mandy
Lawson
,
Andrew S.
Lister
,
David
Longmire
,
Stacey
Marden
,
Thomas M.
Mcguire
,
Caroline
Mcmillan
,
Lindsay
Mcmurray
,
Christopher J.
Morrow
,
J. Willem M.
Nissink
,
Thomas A.
Moss
,
Daniel H.
O’donovan
,
Radoslaw
Polanski
,
Stephen
Stokes
,
Kumar
Thakur
,
Dawn
Trueman
,
Caroline
Truman
,
Michael J.
Tucker
,
Haixia
Wang
,
Nicky
Whalley
,
Dedong
Wu
,
Ye
Wu
,
Bin
Yang
,
Wenzhan
Yang
Diamond Proposal Number(s):
[20015]
Abstract: Herein, we report the optimization of a meta-substituted series of selective estrogen receptor degrader (SERD) antagonists for the treatment of ER+ breast cancer. Structure-based design together with the use of modeling and NMR to favor the bioactive conformation led to a highly potent series of basic SERDs with promising physicochemical properties. Issues with hERG activity resulted in a strategy of zwitterion formation and ultimately in the identification of 38. This compound was shown to be a highly potent SERD capable of effectively degrading ERα in both MCF-7 and CAMA-1 cell lines. The low lipophilicity and zwitterionic nature led to a SERD with a clean secondary pharmacology profile and no hERG activity. Favorable physicochemical properties resulted in good oral bioavailability in preclinical species and potent in vivo activity in a mouse xenograft model.
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Feb 2023
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I03-Macromolecular Crystallography
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Gareth G.
Doherty
,
Geraldine Jia Ming
Ler
,
Norbert
Wimmer
,
Paul V.
Bernhardt
,
Roger A.
Ashmus
,
David J.
Vocadlo
,
Zachary
Armstrong
,
Gideon J.
Davies
,
Mauro
Maccarrone
,
Jin-Ping
Li
,
Yasmin
Kayal
,
Vito
Ferro
Diamond Proposal Number(s):
[24948]
Open Access
Abstract: 1-Azasugar analogues of l-iduronic acid (l-IdoA) and d-glucuronic acid (d-GlcA) and their corresponding enantiomers have been synthesized as potential pharmacological chaperones for mucopolysaccharidosis I (MPS I), a lysosomal storage disease caused by mutations in the gene encoding α-iduronidase (IDUA). The compounds were efficiently synthesized in nine or ten steps from d- or l-arabinose, and the structures were confirmed by X-ray crystallographic analysis of key intermediates. All compounds were inactive against IDUA, although l-IdoA-configured 8 moderately inhibited β-glucuronidase (β-GLU). The d-GlcA-configured 9 was a potent inhibitor of β-GLU and a moderate inhibitor of the endo-β-glucuronidase heparanase. Co-crystallization of 9 with heparanase revealed that the endocyclic nitrogen of 9 forms close interactions with both the catalytic acid and catalytic nucleophile.
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Feb 2023
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I03-Macromolecular Crystallography
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Xiao
Liu
,
Raphael
Reinbold
,
Shuang
Liu
,
Ryan A.
Herold
,
Patrick
Rabe
,
Stéphanie
Duclos
,
Rahul B.
Yadav
,
Martine I.
Abboud
,
Sandrine
Thieffine
,
Fraser A.
Armstrong
,
Lennart
Brewitz
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[23459]
Open Access
Abstract: Variants of isocitrate dehydrogenase (IDH) 1 and 2 (IDH1/2) alter metabolism in cancer cells by catalyzing the NADPH-dependent reduction of 2-oxoglutate (2OG) to (2R)-hydroxyglutarate (2HG). However, it is unclear how derivatives of 2OG can affect cancer cell metabolism. Here, we used synthetic C3 and C4 alkylated 2OG derivatives to investigate the substrate selectivities of the most common cancer-associated IDH1 variant (R132H IDH1), of two cancer-associated IDH2 variants (R172K IDH2, R140Q IDH2), and of wildtype IDH1/2. Absorbance-based, NMR and electrochemical assays were employed to monitor wildtype IDH1/2 and IDH1/2 variant-catalyzed 2OG derivative turnover in the presence and absence of 2OG. Our results reveal that 2OG derivatives can serve as substrates of the investigated IDH1/2 variants, but not of wildtype IDH1/2, and have the potential to act as 2OG-competitive inhibitors. Kinetic parameters reveal that some 2OG derivatives, including the natural product 3-methyl-2OG, are equally or even more efficient IDH1/2 variant substrates compared to 2OG. Furthermore, NMR and mass spectrometry studies confirmed IDH1/2 variant-catalyzed production of alcohols in the cases of the 3-methyl-, 3-butyl-, and 3-benzyl-substituted 2OG derivatives; a crystal structure of 3-butyl-2OG with an IDH1 variant (R132C/S280F IDH1) reveals active site binding. The combined results highlight the potential for (i) IDH1/2 variant-catalyzed reduction of 2-oxoacids other than 2OG in cells, (ii) modulation of IDH1/2 variant activity by 2-oxoacid natural products, including some present in common foods, (iii) inhibition of IDH1/2 variants via active site binding rather than the established allosteric mode of inhibition, and (iv) possible use of IDH1/2 variants as biocatalysts.
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Jan 2023
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B21-High Throughput SAXS
I04-Macromolecular Crystallography
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Open Access
Abstract: Spalt-like 4 (SALL4) maintains vertebrate embryonic stem cell identity and is required for the development of multiple organs, including limbs. Mutations in SALL4 are associated with Okihiro syndrome, and SALL4 is also a known target of thalidomide. SALL4 protein has a distinct preference for AT-rich sequences, recognised by a pair of zinc fingers at the C-terminus. However, unlike many characterised zinc finger proteins, SALL4 shows flexible recognition with many different combinations of AT-rich sequences being targeted. SALL4 interacts with the NuRD corepressor complex which potentially mediates repression of AT-rich genes. We present a crystal structure of SALL4 C-terminal zinc fingers with an AT-rich DNA sequence, which shows that SALL4 uses small hydrophobic and polar side chains to provide flexible recognition in the major groove. Missense mutations reported in patients that lie within the C-terminal zinc fingers reduced overall binding to DNA but not the preference for AT-rich sequences. Furthermore, these mutations altered association of SALL4 with AT-rich genomic sites, providing evidence that these mutations are likely pathogenic.
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Jan 2023
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Krios I-Titan Krios I at Diamond
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Yang
Yang
,
Wenjuan
Zhang
,
Alexey G.
Murzin
,
Manuel
Schweighauser
,
Melissa
Huang
,
Sofia
Lovestam
,
Sew Y.
Peak-Chew
,
Takashi
Saito
,
Takaomi C.
Saido
,
Jennifer
Macdonald
,
Isabelle
Lavenir
,
Bernardino
Ghetti
,
Caroline
Graff
,
Amit
Kumar
,
Agneta
Nordberg
,
Michel
Goedert
,
Sjors H. W.
Scheres
Diamond Proposal Number(s):
[23268]
Open Access
Abstract: The Arctic mutation, encoding E693G in the amyloid precursor protein (APP) gene [E22G in amyloid-β (Aβ)], causes dominantly inherited Alzheimer’s disease. Here, we report the high-resolution cryo-EM structures of Aβ filaments from the frontal cortex of a previously described case (AβPParc1) with the Arctic mutation. Most filaments consist of two pairs of non-identical protofilaments that comprise residues V12–V40 (human Arctic fold A) and E11–G37 (human Arctic fold B). They have a substructure (residues F20–G37) in common with the folds of type I and type II Aβ42. When compared to the structures of wild-type Aβ42 filaments, there are subtle conformational changes in the human Arctic folds, because of the lack of a side chain at G22, which may strengthen hydrogen bonding between mutant Aβ molecules and promote filament formation. A minority of Aβ42 filaments of type II was also present, as were tau paired helical filaments. In addition, we report the cryo-EM structures of Aβ filaments with the Arctic mutation from mouse knock-in line AppNL−G−F. Most filaments are made of two identical mutant protofilaments that extend from D1 to G37 (AppNL−G−F murine Arctic fold). In a minority of filaments, two dimeric folds pack against each other in an anti-parallel fashion. The AppNL−G−F murine Arctic fold differs from the human Arctic folds, but shares some substructure.
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Jan 2023
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