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49 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4, 5 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Structural basis for inhibition of human primase by arabinofuranosyl nucleoside analogues fludarabine and vidarabine Sandro Holzer , Neil J. Rzechorzek , Isobel R. Short , Michael Jenkyn-bedford , Luca Pellegrini , Mairi Kilkenny Acs Chemical Biology DOI: 10.1021/acschembio.9b00367 Diamond Proposal Number(s): [9537] Show Abstract ▼ Abstract: Nucleoside analogues are widely used in clinical practice as chemotherapy drugs. Arabinose nucleoside derivatives such as fludarabine are effective in the treatment of patients with acute and chronic leukemias and non-Hodgkin lymphomas. Although nucleoside analogues are generally known to function by inhibiting DNA synthesis in rapidly proliferating cells, the identity of their in vivo targets and mechanism of action are often not known in molecular detail. Here we provide a structural basis for arabinose nucleotide-mediated inhibition of human primase, the DNA-dependent RNA polymerase responsible for initiation of DNA synthesis in DNA replication. Our data suggest ways in which the chemical structure of fludarabine could be modified to improve its specificity and affinity towards primase, possibly leading to less toxic and more effective therapeutic agents.	Sep 2019
B21-High Throughput SAXS	Double monoubiquitination modifies the molecular recognition properties of p15 PAF promoting binding to the reader module of Dnmt1 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 Acs Chemical Biology DOI: 10.1021/acschembio.9b00679 Diamond Proposal Number(s): [14707] Show Abstract ▼ 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.	Sep 2019
I04-1-Macromolecular Crystallography (fixed wavelength)	Rapid identification of novel allosteric PRC2 inhibitors Jon A. Read , Jonathan Tart , Philip B. Rawlins , Clare Gregson , Karen Jones , Ning Gao , Xiahui Zhu , Ron Tomlinson , Erin Code , Tony Cheung , Huawei Chen , Sameer P. Kawatkar , Andy Bloecher , Sharan Bagal , Daniel H. O’donovan , James Robinson Acs Chemical Biology DOI: 10.1021/acschembio.9b00468 Show Abstract ▼ Abstract: Enhancer of zeste homologue 2 (EZH2), the catalytic subunit of polycomb repressive complex 2 (PRC2), regulates chromatin state and gene expression by methylating histone H3 lysine 27. EZH2 is overexpressed or mutated in various hematological malignancies and solid cancers. Our previous efforts to identify inhibitors of PRC2 methyltransferase activity by high-throughput screening (HTS) resulted in large numbers of false positives and thus a significant hit deconvolution challenge. More recently, others have reported compounds that bind to another PRC2 core subunit, EED, and allosterically inhibit EZH2 activity. This mechanism is particularly appealing as it appears to retain potency in cell lines that have acquired resistance to orthosteric EZH2 inhibition. By designing a fluorescence polarization probe based on the reported EED binding compounds, we were able to quickly and cleanly re-triage our previously challenging HTS hit list and identify novel allosteric PRC2 inhibitors.	Sep 2019
I04-1-Macromolecular Crystallography (fixed wavelength)	Structural basis of glycerophosphodiester recognition by the Mycobacterium tuberculosis substrate-binding protein UgpB Jonathan S. Fenn , Ridvan Nepravishta , Collette S. Guy , James Harrison , Jesus Angulo , Alexander D. Cameron , Elizabeth Fullam Acs Chemical Biology DOI: 10.1021/acschembio.9b00204 Diamond Proposal Number(s): [14692] Show Abstract ▼ Abstract: Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB) and has evolved an incredible ability to survive latently within the human host for decades. The Mtb pathogen encodes for a low number of ATP-binding cassette (ABC) importers for the acquisition of carbohydrates that may reflect the nutrient poor environment within the host macrophages. Mtb UgpB (Rv2833c) is the substrate binding domain of the UgpABCE transporter that recognizes glycerophosphocholine (GPC), indicating that this transporter has a role in recycling glycerophospholipid metabolites. By using a combination of saturation transfer difference (STD) NMR and X-ray crystallography, we report the structural analysis of Mtb UgpB complexed with GPC and have identified that Mtb UgpB not only recognizes GPC but is also promiscuous for a broad range of glycerophosphodiesters. Complementary biochemical analyses and site-directed mutagenesis precisely define the molecular basis and specificity of glycerophosphodiester recognition. Our results provide critical insights into the structural and functional role of the Mtb UgpB transporter and reveal that the specificity of this ABC-transporter is not limited to GPC, therefore optimizing the ability of Mtb to scavenge scarce nutrients and essential glycerophospholipid metabolites via a single transporter during intracellular infection.	Aug 2019
I02-Macromolecular Crystallography	Fluorescent retinoic acid analogues as probes for biochemical and intracellular characterization of retinoid signaling pathways 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 Acs Chemical Biology DOI: 10.1021/acschembio.8b00916 Show Abstract ▼ 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.	Feb 2019
I03-Macromolecular Crystallography	Sphingobacterium sp. T2 manganese superoxide dismutase catalyses the oxidative demethylation of polymeric lignin via generation of hydroxyl radical Goran M. M. Rashid , Xiaoyang Zhang , Rachael C. Wilkinson , Vilmos Fulop , Betty Cottyn , Stéphanie Baumberger , Timothy D. H. Bugg Acs Chemical Biology DOI: 10.1021/acschembio.8b00557 Diamond Proposal Number(s): [14692] Show Abstract ▼ Abstract: Sphingobacterium sp. T2 contains two extracellular manganese superoxide dismutase enzymes which exhibit unprecedented activity for lignin oxidation, but via an unknown mechanism. Enzymatic treatment of lignin model compounds gave products whose structures were indicative of aryl-C oxidative cleavage and demethylation, as well as alkene dihydroxylation and alcohol oxidation. 18O labelling studies on the SpMnSOD-catalysed oxidation of lignin model compound guiaiacylglycerol--guaiacyl ether indicated that the oxygen atom inserted by the enzyme is derived from superoxide or peroxide. Analysis of an alkali lignin treated by SpMnSOD1 by quantitative 31P NMR spectroscopy demonstrated 20-40% increases in phenolic and aliphatic OH content, consistent with lignin demethylation and some internal oxidative cleavage reactions. Assay for hydroxyl radical generation using a fluorometric hydroxyphenylfluorescein assay revealed the release of approximately 1 molar equivalent of hydroxyl radical by SpMnSOD1. Four amino acid replacements in SpMnSOD1 were investigated, and A31H or Y27H site-directed mutant enzymes were found to show no lignin demethylation activity according to 31P NMR analysis. Structure determination of the A31H and Y27H mutant enzymes reveals the repositioning of an N-terminal protein loop, leading to widening of a solvent channel at the dimer interface, which would provide increased solvent access to the Mn centre for hydroxyl radical generation.	Sep 2018
I04-Macromolecular Crystallography	Type II kinase inhibitors targeting Cys-gatekeeper kinases display orthogonality with wild type and Ala/Gly-gatekeeper kinases Cory A. Ocasio , Alexander A. Warkentin , Patrick J. Mcintyre , Krister J. Barkovich , Clare Vesely , John Spencer , Kevan M. Shokat , Richard Bayliss Acs Chemical Biology DOI: 10.1021/acschembio.8b00592 Show Abstract ▼ Abstract: Analog-sensitive (AS) kinases contain large to small mutations in the gatekeeper position rendering them susceptible to inhibition with bulky analogs of pyrazolopyrimidine-based Src kinase inhibitors (e.g. PP1). This ‘bump-hole’ method has been utilized for at least 85 of ~520 kinases, but many kinases are intolerant to this approach. To expand the scope of AS-kinase technology, we designed type II kinase inhibitors, ASDO2/6 (Analog-Sensitive ‘DFG-Out’ kinase inhibitors-2/6), that target the ‘DFG-out’ conformation of cysteine (Cys)-gatekeeper kinases with submicromolar potency. We validated this system in vitro against Greatwall kinase (GWL), Aurora-A kinase and Cyclin-dependent kinase-1 and in cells using M110C-GWL expressing mouse embryonic fibroblasts. These Cys-gatekeeper kinases were sensitive to ASDO2/6-inhibition, but not AS-kinase inhibitor 3MB-PP1 and vice versa. These compounds, with AS-kinase inhibitors, have the potential to inhibit multiple AS-kinases independently with applications in systems level and translational kinase research as well as the rational design of type II kinase inhibitors targeting endogenous kinases.	Sep 2018
I24-Microfocus Macromolecular Crystallography	A key role for the periplasmic PfeE esterase in iron acquisition via the siderophore Enterobactin in Pseudomonas aeruginosa Quentin Perraud , Lucile Moynie , Véronique Gasser , Mathilde Munier , Julien Godet , Françoise Hoegy , Yves Mély , Gaëtan L. A. Mislin , James H. Naismith , Isabelle J. Schalk Acs Chemical Biology DOI: 10.1021/acschembio.8b00543 Show Abstract ▼ Abstract: Enterobactin (ENT) is a siderophore (iron-chelating compound) produced by Escherichia coli in order to gain access to iron, an essential nutriment for bacterial growth. ENT is used as an exosiderophore by the opportunistic human pathogen Pseudomonas aeruginosa with transport of ferri-ENT across the bacterial outer membrane by the transporter PfeA. Next to pfeA gene on the chromosome is localized a gene encoding for an esterase, PfeE, whose transcription is regulated, as for pfeA, by the presence of ENT in bacterial environment. Purified PfeE hydrolyzed ferri-ENT into three molecules of 2,3 DHBS (2,3 dihydroxybenzoylserine) still complexed with ferric iron, and complete dissociation of iron from ENT chelating groups was only possible in the presence of both PfeE and an iron reducer, such as DTT. The crystal structure of PfeE and an inactive PfeE mutant complexed with ferri-ENT or a non-hydrolysable ferri-catechol complex allowed identification of the enzyme binding site and the catalytic triad. Finally, cell fractionation and fluorescence microscopy showed periplasmic localization of PfeE in P. aeruginosa cells. Thus, the molecular mechanism of iron release from ENT in P. aeruginosa differs from that previously described in E. coli. In P. aeruginosa, siderophore hydrolysis occurs in the periplasm, with ENT never reaching the bacterial cytoplasm. In E. coli, ferri-ENT crosses the inner membrane via the ABC transporter FepBCD and ferri-ENT is hydrolyzed by the esterase Fes only once it is in the cytoplasm.	Aug 2018
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Targeting ligandable pockets on PHD zinc finger domains by a fragment-based approach Anastasia Amato , Xavier Lucas , Alessio Bortoluzzi , David Wright , Alessio Ciulli Acs Chemical Biology DOI: 10.1021/acschembio.7b01093 Diamond Proposal Number(s): [14980, 10071] Show Abstract ▼ Abstract: Plant homeodomain (PHD) zinc fingers are histone reader domains that often associate with human diseases. Despite this, they constitute a poorly targeted class of readers, suggesting low ligandability. Here, we describe a successful fragment-based campaign targeting PHD fingers from the proteins BAZ2A and BAZ2B as model systems. We validated a pool of in silico fragments both biophysically and structurally and solved the first crystal structures of PHD zinc fingers in complex with fragments bound to an anchoring pocket at the histone binding site. The best-validated hits were found to displace a histone H3 tail peptide in competition assays. This work identifies new chemical scaffolds that provide suitable starting points for future ligand optimization using structure-guided approaches. The demonstrated ligandability of the PHD reader domains could pave the way for the development of chemical probes to drug this family of epigenetic targets.	Mar 2018
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Characterization of the fast and promiscuous macrocyclase from plant PCY1 enables the use of simple substrates Hannes Ludewig , Clarissa M. Czekster , Emilia Oueis , Elizabeth S. Munday , Mohammed Arshad , Silvia A. Synowsky , Andrew F. Bent , James H. Naismith Acs Chemical Biology DOI: 10.1021/acschembio.8b00050 Show Abstract ▼ Abstract: Cyclic ribosomally derived peptides possess diverse bioactivities and are currently of major interest in drug development. However, it can be chemically challenging to synthesize these molecules, hindering the diversification and testing of cyclic peptide leads. Enzymes used in vitro offer a solution to this, however peptide macrocyclization remains the bottleneck. PCY1, involved in the biosynthesis of plant orbitides, belongs to the class of prolyl oligopeptidases and natively displays substrate promiscuity. PCY1 is a promising candidate for in vitro utilization but its substrates require an 11 to 16 residue C-terminal recognition tail. We have characterized PCY1 both kinetically and structurally with multiple substrate complexes revealing for the molecular basis of recognition and catalysis. Using these insights, we have identified a three residue C-terminal extension that replaces the natural recognition tail permitting PCY1 to operate on synthetic substrates. We demonstrate that PCY1 can macrocyclize a variety of substrates with this short tail, including unnatural amino acids and non-amino acids, highlighting PCY1's potential in biocatalysis.	Jan 2018

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