B21-High Throughput SAXS
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Amaia
Gonzalez-Magaña
,
Alain
Ibáñez De Opakua
,
Nekane
Merino
,
Hugo
Monteiro
,
Tammo
Diercks
,
Javier
Murciano-Calles
,
Irene
Luque
,
Pau
Bernadó
,
Tiago
Cordeiro
,
Alfredo
De Biasio
,
Francisco J
Blanco
Diamond Proposal Number(s):
[14707]
Open Access
Abstract: The Proliferating Cell Nuclear Antigen-associated factor p15PAF is a nuclear protein that acts as a regulator of DNA repair during DNA replication. The p15PAF gene is overexpressed in several types of human cancer and its function is regulated by monoubiquitination of two lysines (K15 and K24) at the protein N-terminal region. We have previously shown that p15PAF is an intrinsically disordered protein which partially folds upon binding to PCNA and independently contacts DNA through its N-terminal tail. Here we present an NMR conformational characterization of p15PAF monoubiquitinated at both K15 and K24 via a disulfide bridge mimicking the isopeptide bond. We show that doubly monoubiquitinated p15PAF is monomeric, intrinsically disordered, binds to PCNA as non-ubiquitinated p15PAF does, but interacts with DNA with reduced affinity. Our SAXS-derived conformational ensemble of doubly monoubiquitinated p15PAF shows that the ubiquitin moieties, separated by 8 disordered residues, form transient dimers due to the high local effective concentration. This observation and the sequence similarity with histone H3 N-terminal tail suggest that doubly monoubiquitinated p15PAF is a binding target of DNA methyl transferase Dnmt1, as confirmed by calorimetry. Therefore, doubly monoubiquitinated p15PAF directly interacts with PCNA and recruits Dnmt1for maintenance of DNA methylation during replication.
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Sep 2019
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7613]
Open Access
Abstract: DNA transposases catalyze the movement of transposons around genomes by a cut-and-paste mechanism related to retroviral integration. Transposases and retroviral integrases share a common RNaseH-like domain with a catalytic DDE/D triad that coordinates the divalent cations required for DNA cleavage and integration. The anti-retroviral drugs Raltegravir and Elvitegravir inhibit integrases by displacing viral DNA ends from the catalytic metal ions. We demonstrate that Raltegravir, but not Elvitegravir, binds to Mos1 transposase in the presence of Mg2+ or Mn2+, without the requirement for transposon DNA, and inhibits transposon cleavage and DNA integration in biochemical assays. Crystal structures at 1.7 Å resolution show Raltegravir, in common with integrases, coordinating two Mg2+ or Mn2+ ions in the Mos1 active site. However, in the absence of transposon ends, the drug adopts an unusual, compact binding mode distinct from that observed in the active site of the prototype foamy virus integrase.
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Jan 2014
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I02-Macromolecular Crystallography
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David
Albesa-Jove
,
Laurent R.
Chiarelli
,
Vadim
Makarov
,
Maria Rosalia
Pasca
,
Saioa
Urresti
,
Giorgia
Mori
,
Elena
Salina
,
Anthony
Vocat
,
Natalia
Comino
,
Elisabeth
Mohorko
,
Svetlana
Ryabova
,
Bernhard
Pfieiffer
,
Ana Luisa De Jesus
Lopes Ribeiro
,
Ane
Rodrigo-Unzueta
,
Montse
Tersa
,
Giuseppe
Zanoni
,
Silvia
Buroni
,
Karl-Heinz
Altmann
,
Ruben C.
Hartkoorn
,
Rudi
Glockshuber
Diamond Proposal Number(s):
[8302]
Abstract: The emergence of multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis highlights the need to discover new antitubercular agents. Here we describe the synthesis and characterization of a new series of thienopyrimidine (TP) compounds that kill both replicating and non-replicating M. tuberculosis. The strategy to determine the mechanism of action of these TP derivatives was to generate resistant mutants to the most effective compound TP053 and to isolate the genetic mutation responsible for this phenotype. The only non-synonymous mutation found was a g83c transition in the Rv2466c gene, resulting in the replacement of tryptophan 28 by a serine. The Rv2466c overexpression increased the sensitivity of M. tuberculosis wild-type and resistant mutant strains to TP053, indicating that TP053 is a prodrug activated by Rv2466c. Biochemical studies performed with purified Rv2466c demonstrated that only the reduced form of Rv2466c can activate TP053. The 1.7 angstrom resolution crystal structure of the reduced form of Rv2466c, a protein whose expression is transcriptionally regulated during the oxidative stress response, revealed a unique homodimer in which a beta-strand is swapped between the thioredoxin domains of each subunit. A pronounced groove harboring the unusual active-site motif CPWC might account for the uncommon reactivity profile of the protein. The mutation of Trp28Ser clearly predicts structural defects in the thioredoxin fold, including the destabilization of the dimerization core and the CPWC motif, likely impairing the activity of Rv2466c against TP053. Altogether our experimental data provide insights into the molecular mechanism underlying the anti- mycobacterial activity of TP-based compounds, paving the way for future drug development programmes.
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Jul 2014
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8367]
Open Access
Abstract: N-Acetylneuraminic acid lyase (NAL) is a Class I aldolase that catalyzes the reversible condensation of pyruvate with N-acetyl-d-mannosamine (ManNAc) to yield the sialic acid N-acetylneuraminic acid (Neu5Ac). Aldolases are finding increasing use as biocatalysts for the stereospecific synthesis of complex molecules. Incomplete understanding of the mechanism of catalysis in aldolases, however, can hamper development of new enzyme activities and specificities, including control over newly generated stereocenters. In the case of NAL, it is clear that the enzyme catalyzes a Bi-Uni ordered condensation reaction in which pyruvate binds first to the enzyme to form a catalytically important Schiff base. The identity of the residues required for catalysis of the condensation step and the nature of the transition state for this reaction, however, have been a matter of conjecture. In order to address, this we crystallized a Y137A variant of the E. coli NAL in the presence of Neu5Ac. The three-dimensional structure shows a full length sialic acid bound in the active site of subunits A, B, and D, while in subunit C, discontinuous electron density reveals the positions of enzyme-bound pyruvate and ManNAc. These ‘snapshot’ structures, representative of intermediates in the enzyme catalytic cycle, provided an ideal starting point for QM/MM modeling of the enzymic reaction of carbon–carbon bond formation. This revealed that Tyr137 acts as the proton donor to the aldehyde oxygen of ManNAc during the reaction, the activation barrier is dominated by carbon–carbon bond formation, and proton transfer from Tyr137 is required to obtain a stable Neu5Ac-Lys165 Schiff base complex. The results also suggested that a triad of residues, Tyr137, Ser47, and Tyr110 from a neighboring subunit, are required to correctly position Tyr137 for its function, and this was confirmed by site-directed mutagenesis. This understanding of the mechanism and geometry of the transition states along the C–C bond-forming pathway will allow further development of these enzymes for stereospecific synthesis of new enzyme products.
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Apr 2014
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I02-Macromolecular Crystallography
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Rasheduzzaman
Chowdhury
,
José Ignacio
Candela-Lena
,
Mun Chiang
Chan
,
David Jeremy
Greenald
,
Kar Kheng
Yeoh
,
Ya-Min
Tian
,
Michael A.
Mcdonough
,
Anthony
Tumber
,
Nathan R.
Rose
,
Ana
Conejo-Garcia
,
Marina
Demetriades
,
Sinnakaruppan
Mathavan
,
Akane
Kawamura
,
Myung Kyu
Lee
,
Freek
Van Eeden
,
Christopher W.
Pugh
,
Peter J.
Ratcliffe
,
Christopher J.
Schofield
Abstract: The hypoxia inducible factor (HIF) system is central to the signaling of low oxygen (hypoxia) in animals. The levels of HIF-α isoforms are regulated in an oxygen-dependent manner by the activity of the HIF prolyl-hydroxylases (PHD or EGLN enzymes), which are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases. Here, we describe biochemical, crystallographic, cellular profiling, and animal studies on PHD inhibitors including selectivity studies using a representative set of human 2OG oxygenases. We identify suitable probe compounds for use in studies on thefunctional effects of PHD inhibition in cells and in animals.
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May 2013
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I02-Macromolecular Crystallography
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Hyung-Gu
Kim
,
Li
Tan
,
Ellen L.
Weisberg
,
Feiyang
Liu
,
Peter
Canning
,
Hwan Geun
Choi
,
Scott A.
Ezell
,
Hong
Wu
,
Zheng
Zhao
,
Jinhua
Wang
,
Anna
Mandinova
,
James D.
Griffin
,
Alex N.
Bullock
,
Qingsong
Liu
,
Sam W.
Lee
,
Nathanael S.
Gray
Open Access
Abstract: The DDR1 receptor tyrosine kinase is activated by matrix collagens and has been implicated in numerous cellular functions such as proliferation, differentiation, adhesion, migration, and invasion. Here we report the discovery of a potent and selective DDR1 inhibitor, DDR1-IN-1, and present the 2.2 Å DDR1 co-crystal structure. DDR1-IN-1 binds to DDR1 in the ‘DFG-out’ conformation and inhibits DDR1 autophosphorylation in cells at submicromolar concentrations with good selectivity as assessed against a panel of 451 kinases measured using the KinomeScan technology. We identified a mutation in the hinge region of DDR1, G707A, that confers >20-fold resistance to the ability of DDR1-IN-1 to inhibit DDR1 autophosphorylation and can be used to establish what pharmacology is DDR1-dependent. A combinatorial screen of DDR1-IN-1 with a library of annotated kinase inhibitors revealed that inhibitors of PI3K and mTOR such as GSK2126458 potentiate the antiproliferative activity of DDR1-IN-1 in colorectal cancer cell lines. DDR1-IN-1 provides a useful pharmacological probe for DDR1-dependent signal transduction.
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Oct 2013
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I02-Macromolecular Crystallography
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Dominic I.
James
,
Kate M.
Smith
,
Allan M.
Jordan
,
Emma E.
Fairweather
,
Louise A.
Griffiths
,
Nicola S.
Hamilton
,
James R.
Hitchin
,
Colin P.
Hutton
,
Stuart
Jones
,
Paul
Kelly
,
Alison E.
Mcgonagle
,
Helen
Small
,
Alexandra I. J.
Stowell
,
Julie
Tucker
,
Ian D.
Waddell
,
Bohdan
Waszkowycz
,
Donald J.
Ogilvie
Open Access
Abstract: The enzyme poly(ADP-ribose) glycohydrolase (PARG) performs a critical role in the repair of DNA single strand breaks (SSBs). However, a detailed understanding of its mechanism of action has been hampered by a lack of credible, cell-active chemical probes. Herein, we demonstrate inhibition of PARG with a small molecule, leading to poly(ADP-ribose) (PAR) chain persistence in intact cells. Moreover, we describe two advanced, and chemically distinct, cell-active tool compounds with convincing on-target pharmacology and selectivity. Using one of these tool compounds, we demonstrate pharmacology consistent with PARG inhibition. Further, while the roles of PARG and poly(ADP-ribose) polymerase (PARP) are closely intertwined, we demonstrate that the pharmacology of a PARG inhibitor differs from that observed with the more thoroughly studied PARP inhibitor olaparib. We believe that these tools will facilitate a wider understanding of this important component of DNA repair and may enable the development of novel therapeutic agents exploiting the critical dependence of tumors on the DNA damage response (DDR).
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Nov 2016
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8922]
Abstract: The oncogenic transcription factor activator protein-1 (AP-1) is a DNA-binding protein that assembles through dimerization of Fos and Jun protein subunits, their leucine-rich helical sequences entwining into a coiled-coil structure. This study reports on downsizing the proto-oncogene cFos protein (380 residues) to shorter peptides (37–25 residues) modified with helix-inducing constraints to enhance binding to Jun. A crystal structure is reported for a 37-residue Fos-derived peptide (FosW) bound to Jun. This guided iterative downsizing of FosW to shorter peptide sequences that were constrained into stable water-soluble α-helices by connecting amino acid side chains to form cyclic pentapeptide components. Structural integrity in the presence and absence of Jun was assessed by circular dichroism spectroscopy, while the thermodynamics of binding to cFos was measured by isothermal titration calorimetry. A 25-residue constrained peptide, one-third shorter yet 25% more helical than the structurally characterized 37-residue Fos-derived peptide, retained 80% of the binding free energy as a result of preorganization in a Jun-binding helix conformation, with the entropy gain (TΔS = +3.2 kcal/mol) compensating for the enthalpy loss. Attaching a cell-penetrating peptide (TAT48–57) and a nuclear localization signal (SV40) promoted cell uptake, localization to the nucleus, and inhibition of the proliferation of two breast cancer cell lines.
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Jun 2017
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I02-Macromolecular Crystallography
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Marion
Schuller
,
Kerstin
Riedel
,
Ian
Gibbs-Seymour
,
Kristin
Uth
,
Christian
Sieg
,
André P
Gehring
,
Ivan
Ahel
,
Franz
Bracher
,
Benedikt M.
Kessler
,
Jonathan M.
Elkins
,
Stefan
Knapp
Abstract: Macrodomains are conserved protein interaction modules that can be found in all domains of life as well as in certain viruses. Macrodomains mediate recognition of sequence motifs harbouring adenosine diphosphate ribose (ADPR) modifications, thereby regulating a variety of cellular processes. Due to their role in cancer or viral pathogenesis, macrodomains have emerged as potential therapeutic targets, but the unavailability of small molecule inhibitors has hampered target validation studies so far. Here, we describe an efficient screening strategy for identification of small molecule inhibitors that displace ADPR from macrodomains. We report the discovery and characterisation of a macrodomain inhibitor, GeA-69, selectively targeting macrodomain 2 (MD2) of PARP14 with low micromolar affinity. Co-crystallisation of a GeA-69 analogue with PARP14 MD2 revealed an allosteric binding mechanism explaining its selectivity over other human macrodomains. We show that GeA-69 engages PARP14 MD2 in intact cells and prevents its localisation to sites of DNA damage.
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Oct 2017
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I02-Macromolecular Crystallography
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David R.
Chisholm
,
Charles W. E.
Tomlinson
,
Garr-Layy
Zhou
,
Claire
Holden
,
Valerie
Affleck
,
Rebecca
Lamb
,
Katherine
Newling
,
Peter
Ashton
,
Roy
Valentine
,
Christopher
Redfern
,
János
Erostyák
,
Geza
Makkai
,
Carrie A.
Ambler
,
Andrew
Whiting
,
Ehmke
Pohl
Open Access
Abstract: Retinoids, such as all-trans-retinoic acid (ATRA), are endogenous signalling molecules derived from Vitamin A that influ-ence a variety of cellular processes through mediation of transcription events in the cell nucleus. Due to these wide-ranging and powerful biological activities, retinoids have emerged as therapeutic candidates of enormous potential. However, their use has been limited, to date, due to a lack of understanding of the complex and intricate signaling pathways that they con-trol. We have designed and synthesized a family of synthetic retinoids that exhibit strong, intrinsic, solvatochromatic fluo-rescence as multifunctional tools to interrogate these important biological activities. We utilized the unique photophysical characteristics of these fluorescent retinoids to develop a novel in vitro fluorometric binding assay to characterize and quanti-fy their binding to their cellular targets, including Cellular Retinoid Binding Protein II (CRABPII). The dihydroquinoline retinoid, DC360, exhibited particularly strong binding (Kd = 34.0 ± 2.5 nM) and we further used X-ray crystallography to solve the structure of the DC360-CRABPII complex to 1.8 Å, which showed that DC360 occupies the known hydrophobic retinoid-binding pocket. Finally, we used confocal fluorescence microscopy to image the cellular behaviour of the com-pounds in cultured human epithelial cells, highlighting a fascinating nuclear localisation, and used RNA sequencing to con-firm that the compounds regulate similar cellular processes to ATRA. We anticipate that the unique properties of these fluo-rescent retinoids can now be used to shed new light on the vital and highly complex retinoid signalling pathway.
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Feb 2019
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