Publications by authors named "Nicola Burgess-Brown"

37 Publications

Structure and activation mechanism of the human liver-type glutaminase GLS2.

Biochimie 2021 Mar 18;185:96-104. Epub 2021 Mar 18.

Brazilian Biosciences National Laboratory, Brazilian Center for Research in Energy and Materials, Campinas, Sao Paulo, Zip Code, 13083-970, Brazil; Sao Carlos Institute of Physics, University of Sao Paulo, Sao Carlos, SP, Zip Code, 13563-120, Brazil. Electronic address:

Cancer cells exhibit an altered metabolic phenotype, consuming higher levels of the amino acid glutamine. This metabolic reprogramming depends on increased mitochondrial glutaminase activity to convert glutamine to glutamate, an essential precursor for bioenergetic and biosynthetic processes in cells. Mammals encode the kidney-type (GLS) and liver-type (GLS2) glutaminase isozymes. GLS is overexpressed in cancer and associated with enhanced malignancy. On the other hand, GLS2 is either a tumor suppressor or an oncogene, depending on the tumor type. The GLS structure and activation mechanism are well known, while the structural determinants for GLS2 activation remain elusive. Here, we describe the structure of the human glutaminase domain of GLS2, followed by the functional characterization of the residues critical for its activity. Increasing concentrations of GLS2 lead to tetramer stabilization, a process enhanced by phosphate. In GLS2, the so-called "lid loop" is in a rigid open conformation, which may be related to its higher affinity for phosphate and lower affinity for glutamine; hence, it has lower glutaminase activity than GLS. The lower affinity of GLS2 for glutamine is also related to its less electropositive catalytic site than GLS, as indicated by a Thr225Lys substitution within the catalytic site decreasing the GLS2 glutamine concentration corresponding to half-maximal velocity (K). Finally, we show that the Lys253Ala substitution (corresponding to the Lys320Ala in the GLS "activation" loop, formerly known as the "gating" loop) renders a highly active protein in stable tetrameric form. We conclude that the "activation" loop, a known target for GLS inhibition, may also be a drug target for GLS2.
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http://dx.doi.org/10.1016/j.biochi.2021.03.009DOI Listing
March 2021

Expression Screening of Human Integral Membrane Proteins Using BacMam.

Methods Mol Biol 2021 ;2199:95-115

Structural Genomics Consortium, University of Oxford, Oxford, UK.

This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the BacMam system. This eukaryotic expression system was selected and a screening process established in 2016 to enable production of highly challenging human integral membrane proteins (IMPs), which are a significant component of our target list. Here, we discuss our recently developed platform for identifying expression and monodispersity of IMPs from 3 mL of HEK293 cells.
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http://dx.doi.org/10.1007/978-1-0716-0892-0_6DOI Listing
March 2021

Screening and Production of Recombinant Human Proteins: Protein Production in Insect Cells.

Methods Mol Biol 2021 ;2199:67-94

Structural Genomics Consortium, University of Oxford, Oxford, UK.

This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the baculovirus expression vector system (BEVS). This eukaryotic expression system was selected and a screening process established in 2007 as a measure to tackle the more challenging kinase, RNA-DNA processing, and integral membrane protein families on our target list. Here, we discuss our platform for identifying soluble proteins from 3 mL of insect cell culture and describe the procedures involved in producing protein from liter-scale cultures.
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http://dx.doi.org/10.1007/978-1-0716-0892-0_5DOI Listing
March 2021

Screening and Production of Recombinant Human Proteins: Protein Production in E. coli.

Methods Mol Biol 2021 ;2199:45-66

Structural Genomics Consortium, Department of Medicine, Solna, Karolinska Institutet, Solna, Sweden.

In Chapter 3 , we described the Structural Genomics Consortium (SGC) process for generating multiple constructs of truncated versions of each protein using LIC. In this chapter we provide a step-by-step procedure of our E. coli system for test expressing intracellular (soluble) proteins in a 96-well format that enables us to identify which proteins or truncated versions are expressed in a soluble and stable form suitable for structural studies. In addition, we detail the process for scaling up cultures for large-scale protein purification. This level of production is required to obtain sufficient quantities (i.e., milligram amounts) of protein for further characterization and/or structural studies (e.g., crystallization or cryo-EM experiments). Our standard process is purification by immobilized metal affinity chromatography (IMAC) using nickel resin followed by size exclusion chromatography (SEC), with additional procedures arising from the complexity of the protein itself.
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http://dx.doi.org/10.1007/978-1-0716-0892-0_4DOI Listing
March 2021

Screening and Production of Recombinant Human Proteins: Ligation-Independent Cloning.

Methods Mol Biol 2021 ;2199:23-43

Structural Genomics Consortium, University of Oxford, Oxford, UK.

Structural genomics groups have identified the need to generate multiple truncated versions of each target to improve their success in producing a well-expressed, soluble, and stable protein and one that crystallizes and diffracts to a sufficient resolution for structural determination. At the Structural Genomics Consortium, we opted for the ligation-independent cloning (LIC) method which provides the throughput we desire to produce and screen many proteins in a parallel process. Here, we describe our LIC protocol for generating constructs in 96-well format and provide a choice of vectors suitable for expressing proteins in both E. coli and the baculovirus expression vector system (BEVS).
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http://dx.doi.org/10.1007/978-1-0716-0892-0_3DOI Listing
March 2021

A lower X-gate in TASK channels traps inhibitors within the vestibule.

Nature 2020 06 29;582(7812):443-447. Epub 2020 Apr 29.

Structural Genomics Consortium, University of Oxford, Oxford, UK.

TWIK-related acid-sensitive potassium (TASK) channels-members of the two pore domain potassium (K) channel family-are found in neurons, cardiomyocytes and vascular smooth muscle cells, where they are involved in the regulation of heart rate, pulmonary artery tone, sleep/wake cycles and responses to volatile anaesthetics. K channels regulate the resting membrane potential, providing background K currents controlled by numerous physiological stimuli. Unlike other K channels, TASK channels are able to bind inhibitors with high affinity, exceptional selectivity and very slow compound washout rates. As such, these channels are attractive drug targets, and TASK-1 inhibitors are currently in clinical trials for obstructive sleep apnoea and atrial fibrillation. In general, potassium channels have an intramembrane vestibule with a selectivity filter situated above and a gate with four parallel helices located below; however, the K channels studied so far all lack a lower gate. Here we present the X-ray crystal structure of TASK-1, and show that it contains a lower gate-which we designate as an 'X-gate'-created by interaction of the two crossed C-terminal M4 transmembrane helices at the vestibule entrance. This structure is formed by six residues (VLRFMT) that are essential for responses to volatile anaesthetics, neurotransmitters and G-protein-coupled receptors. Mutations within the X-gate and the surrounding regions markedly affect both the channel-open probability and the activation of the channel by anaesthetics. Structures of TASK-1 bound to two high-affinity inhibitors show that both compounds bind below the selectivity filter and are trapped in the vestibule by the X-gate, which explains their exceptionally low washout rates. The presence of the X-gate in TASK channels explains many aspects of their physiological and pharmacological behaviour, which will be beneficial for the future development and optimization of TASK modulators for the treatment of heart, lung and sleep disorders.
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http://dx.doi.org/10.1038/s41586-020-2250-8DOI Listing
June 2020

The RESOLUTE consortium: unlocking SLC transporters for drug discovery.

Authors:
Giulio Superti-Furga Daniel Lackner Tabea Wiedmer Alvaro Ingles-Prieto Barbara Barbosa Enrico Girardi Ulrich Goldmann Bettina Gürtl Kristaps Klavins Christoph Klimek Sabrina Lindinger Eva Liñeiro-Retes André C Müller Svenja Onstein Gregor Redinger Daniela Reil Vitaly Sedlyarov Gernot Wolf Matthew Crawford Robert Everley David Hepworth Shenping Liu Stephen Noell Mary Piotrowski Robert Stanton Hui Zhang Salvatore Corallino Andrea Faedo Maria Insidioso Giovanna Maresca Loredana Redaelli Francesca Sassone Lia Scarabottolo Michela Stucchi Paola Tarroni Sara Tremolada Helena Batoulis Andreas Becker Eckhard Bender Yung-Ning Chang Alexander Ehrmann Anke Müller-Fahrnow Vera Pütter Diana Zindel Bradford Hamilton Martin Lenter Diana Santacruz Coralie Viollet Charles Whitehurst Kai Johnsson Philipp Leippe Birgit Baumgarten Lena Chang Yvonne Ibig Martin Pfeifer Jürgen Reinhardt Julian Schönbett Paul Selzer Klaus Seuwen Charles Bettembourg Bruno Biton Jörg Czech Hélène de Foucauld Michel Didier Thomas Licher Vincent Mikol Antje Pommereau Frédéric Puech Veeranagouda Yaligara Aled Edwards Brandon J Bongers Laura H Heitman Ad P IJzerman Huub J Sijben Gerard J P van Westen Justine Grixti Douglas B Kell Farah Mughal Neil Swainston Marina Wright-Muelas Tina Bohstedt Nicola Burgess-Brown Liz Carpenter Katharina Dürr Jesper Hansen Andreea Scacioc Giulia Banci Claire Colas Daniela Digles Gerhard Ecker Barbara Füzi Viktoria Gamsjäger Melanie Grandits Riccardo Martini Florentina Troger Patrick Altermatt Cédric Doucerain Franz Dürrenberger Vania Manolova Anna-Lena Steck Hanna Sundström Maria Wilhelm Claire M Steppan

Nat Rev Drug Discov 2020 07;19(7):429-430

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http://dx.doi.org/10.1038/d41573-020-00056-6DOI Listing
July 2020

The structural basis of lipid scrambling and inactivation in the endoplasmic reticulum scramblase TMEM16K.

Nat Commun 2019 09 2;10(1):3956. Epub 2019 Sep 2.

Structural Genomics Consortium, Nuffield Department of Medicine, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ, UK.

Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum (ER) the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase or chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident lipid scramblase with a requirement for short chain lipids and calcium for robust activity. Crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional cryo-EM structures reveal extensive conformational changes from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity.
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http://dx.doi.org/10.1038/s41467-019-11753-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6718402PMC
September 2019

High-Throughput Site-Directed Mutagenesis.

Methods Mol Biol 2019 ;2025:281-296

Structural Genomics Consortium, University of Oxford, Oxford, UK.

Protein engineering has an array of uses: whether you are studying a disease mutation, removing undesirable sequences, adding stabilizing mutations for structural purposes, or simply dissecting protein function. Protein engineering is almost exclusively performed using site-directed mutagenesis (SDM) as this provides targeted modification of specific amino acids, as well as the option of rewriting the native sequence to include or exclude certain regions. Despite its widespread use, SDM has often proved to be a bottleneck, requiring precision manipulation on a sample-by-sample basis to make it work. When dealing with large volumes of samples it is not possible to use such a low-throughput approach. Here we describe a high-throughput (HTP) method for SDM, optimized and used by the Structural Genomics Consortium (SGC) to complement structural studies.
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http://dx.doi.org/10.1007/978-1-4939-9624-7_13DOI Listing
March 2020

Structures of DPAGT1 Explain Glycosylation Disease Mechanisms and Advance TB Antibiotic Design.

Cell 2018 11;175(4):1045-1058.e16

Structural Genomics Consortium, University of Oxford, Oxford, OX3 7DQ, UK. Electronic address:

Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic "lipid-altered" tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.
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http://dx.doi.org/10.1016/j.cell.2018.10.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218659PMC
November 2018

The SGC beyond structural genomics: redefining the role of 3D structures by coupling genomic stratification with fragment-based discovery.

Essays Biochem 2017 11 8;61(5):495-503. Epub 2017 Nov 8.

Structural Genomics Consortium (SGC), Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7DQ, U.K.

The ongoing explosion in genomics data has long since outpaced the capacity of conventional biochemical methodology to verify the large number of hypotheses that emerge from the analysis of such data. In contrast, it is still a gold-standard for early phenotypic validation towards small-molecule drug discovery to use probe molecules (or tool compounds), notwithstanding the difficulty and cost of generating them. Rational structure-based approaches to ligand discovery have long promised the efficiencies needed to close this divergence; in practice, however, this promise remains largely unfulfilled, for a host of well-rehearsed reasons and despite the huge technical advances spearheaded by the structural genomics initiatives of the noughties. Therefore the current, fourth funding phase of the Structural Genomics Consortium (SGC), building on its extensive experience in structural biology of novel targets and design of protein inhibitors, seeks to redefine what it means to do structural biology for drug discovery. We developed the concept of a Target Enabling Package (TEP) that provides, through reagents, assays and data, the missing link between genetic disease linkage and the development of usefully potent compounds. There are multiple prongs to the ambition: rigorously assessing targets' genetic disease linkages through crowdsourcing to a network of collaborating experts; establishing a systematic approach to generate the protocols and data that comprise each target's TEP; developing new, X-ray-based fragment technologies for generating high quality chemical matter quickly and cheaply; and exploiting a stringently open access model to build multidisciplinary partnerships throughout academia and industry. By learning how to scale these approaches, the SGC aims to make structures finally serve genomics, as originally intended, and demonstrate how 3D structures systematically allow new modes of druggability to be discovered for whole classes of targets.
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http://dx.doi.org/10.1042/EBC20170051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5869235PMC
November 2017

Potent and Selective KDM5 Inhibitor Stops Cellular Demethylation of H3K4me3 at Transcription Start Sites and Proliferation of MM1S Myeloma Cells.

Cell Chem Biol 2017 Mar 2;24(3):371-380. Epub 2017 Mar 2.

Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, UK; Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Oxford OX3 7FZ, UK. Electronic address:

Methylation of lysine residues on histone tail is a dynamic epigenetic modification that plays a key role in chromatin structure and gene regulation. Members of the KDM5 (also known as JARID1) sub-family are 2-oxoglutarate (2-OG) and Fe-dependent oxygenases acting as histone 3 lysine 4 trimethyl (H3K4me3) demethylases, regulating proliferation, stem cell self-renewal, and differentiation. Here we present the characterization of KDOAM-25, an inhibitor of KDM5 enzymes. KDOAM-25 shows biochemical half maximal inhibitory concentration values of <100 nM for KDM5A-D in vitro, high selectivity toward other 2-OG oxygenases sub-families, and no off-target activity on a panel of 55 receptors and enzymes. In human cell assay systems, KDOAM-25 has a half maximal effective concentration of ∼50 μM and good selectivity toward other demethylases. KDM5B is overexpressed in multiple myeloma and negatively correlated with the overall survival. Multiple myeloma MM1S cells treated with KDOAM-25 show increased global H3K4 methylation at transcriptional start sites and impaired proliferation.
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http://dx.doi.org/10.1016/j.chembiol.2017.02.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5361737PMC
March 2017

Structure of the polycystic kidney disease TRP channel Polycystin-2 (PC2).

Nat Struct Mol Biol 2017 02 19;24(2):114-122. Epub 2016 Dec 19.

Structural Genomics Consortium, University of Oxford, Oxford, UK.

Mutations in either polycystin-1 (PC1 or PKD1) or polycystin-2 (PC2, PKD2 or TRPP1) cause autosomal-dominant polycystic kidney disease (ADPKD) through unknown mechanisms. Here we present the structure of human PC2 in a closed conformation, solved by electron cryomicroscopy at 4.2-Å resolution. The structure reveals a novel polycystin-specific 'tetragonal opening for polycystins' (TOP) domain tightly bound to the top of a classic transient receptor potential (TRP) channel structure. The TOP domain is formed from two extensions to the voltage-sensor-like domain (VSLD); it covers the channel's endoplasmic reticulum lumen or extracellular surface and encloses an upper vestibule, above the pore filter, without blocking the ion-conduction pathway. The TOP-domain fold is conserved among the polycystins, including the homologous channel-like region of PC1, and is the site of a cluster of ADPKD-associated missense variants. Extensive contacts among the TOP-domain subunits, the pore and the VSLD provide ample scope for regulation through physical and chemical stimuli.
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http://dx.doi.org/10.1038/nsmb.3343DOI Listing
February 2017

Structural analysis of human KDM5B guides histone demethylase inhibitor development.

Nat Chem Biol 2016 07 23;12(7):539-45. Epub 2016 May 23.

Structural Genomics Consortium, University of Oxford, Headington, UK.

Members of the KDM5 (also known as JARID1) family are 2-oxoglutarate- and Fe(2+)-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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http://dx.doi.org/10.1038/nchembio.2087DOI Listing
July 2016

8-Substituted Pyrido[3,4-d]pyrimidin-4(3H)-one Derivatives As Potent, Cell Permeable, KDM4 (JMJD2) and KDM5 (JARID1) Histone Lysine Demethylase Inhibitors.

J Med Chem 2016 Feb 7;59(4):1388-409. Epub 2016 Jan 7.

Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research , 15 Cotswold Road, London SM2 5NG, U.K.

We report the discovery of N-substituted 4-(pyridin-2-yl)thiazole-2-amine derivatives and their subsequent optimization, guided by structure-based design, to give 8-(1H-pyrazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-ones, a series of potent JmjC histone N-methyl lysine demethylase (KDM) inhibitors which bind to Fe(II) in the active site. Substitution from C4 of the pyrazole moiety allows access to the histone peptide substrate binding site; incorporation of a conformationally constrained 4-phenylpiperidine linker gives derivatives such as 54j and 54k which demonstrate equipotent activity versus the KDM4 (JMJD2) and KDM5 (JARID1) subfamily demethylases, selectivity over representative exemplars of the KDM2, KDM3, and KDM6 subfamilies, cellular permeability in the Caco-2 assay, and, for 54k, inhibition of H3K9Me3 and H3K4Me3 demethylation in a cell-based assay.
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http://dx.doi.org/10.1021/acs.jmedchem.5b01635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4770324PMC
February 2016

Optimisation of a triazolopyridine based histone demethylase inhibitor yields a potent and selective KDM2A (FBXL11) inhibitor.

Medchemcomm 2014 Dec;5(12):1879-1886

Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Headington OX3 7DQ, UK.

A potent inhibitor of the JmjC histone lysine demethylase KDM2A (compound , pIC 7.2) with excellent selectivity over representatives from other KDM subfamilies has been developed; the discovery that a triazolopyridine compound binds to the active site of JmjC KDMs was followed by optimisation of the triazole substituent for KDM2A inhibition and selectivity.
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http://dx.doi.org/10.1039/C4MD00291ADOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4678576PMC
December 2014

Crystal structure of Porphyromonas gingivalis peptidylarginine deiminase: implications for autoimmunity in rheumatoid arthritis.

Ann Rheum Dis 2016 06 24;75(6):1255-61. Epub 2015 Jul 24.

Kennedy institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, UK.

Background: 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.

Objectives: By solving the 3D structure of PPAD we aim to characterise activity and elucidate potential mechanisms involved in breach of tolerance to citrullinated proteins in RA.

Methods: PPAD and a catalytically inactive mutant PPAD(C351A) were crystallised and their 3D structures solved. Key residues identified from 3D structures were examined by mutations. Fibrinogen and α-enolase were incubated with PPAD and P. gingivalis arginine gingipain (RgpB) and citrullinated peptides formed were sequenced and quantified by mass spectrometry.

Results: Here, we solve the crystal structure of a truncated, highly active form of PPAD. We confirm catalysis is mediated by the following residues: Asp130, His236, Asp238, Asn297 and Cys351 and show Arg152 and Arg154 may determine the substrate specificity of PPAD for C-terminal arginines. We demonstrate the formation of 37 C-terminally citrullinated peptides from fibrinogen and 11 from α-enolase following incubation with tPPAD and RgpB.

Conclusions: PPAD displays an unequivocal specificity for C-terminal arginine residues and readily citrullinates peptides from key RA autoantigens. The formation of these novel citrullinated peptides may be involved in breach of tolerance to citrullinated proteins in RA.
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http://dx.doi.org/10.1136/annrheumdis-2015-207656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4893104PMC
June 2016

K2P channel gating mechanisms revealed by structures of TREK-2 and a complex with Prozac.

Science 2015 Mar;347(6227):1256-9

Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, UK. OXION Initiative in Ion Channels and Disease, University of Oxford, Oxford OX1 3PN, UK.

TREK-2 (KCNK10/K2P10), a two-pore domain potassium (K2P) channel, is gated by multiple stimuli such as stretch, fatty acids, and pH and by several drugs. However, the mechanisms that control channel gating are unclear. Here we present crystal structures of the human TREK-2 channel (up to 3.4 angstrom resolution) in two conformations and in complex with norfluoxetine, the active metabolite of fluoxetine (Prozac) and a state-dependent blocker of TREK channels. Norfluoxetine binds within intramembrane fenestrations found in only one of these two conformations. Channel activation by arachidonic acid and mechanical stretch involves conversion between these states through movement of the pore-lining helices. These results provide an explanation for TREK channel mechanosensitivity, regulation by diverse stimuli, and possible off-target effects of the serotonin reuptake inhibitor Prozac.
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http://dx.doi.org/10.1126/science.1261512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6034649PMC
March 2015

Assessing cellular efficacy of bromodomain inhibitors using fluorescence recovery after photobleaching.

Epigenetics Chromatin 2014 13;7:14. Epub 2014 Jul 13.

Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK ; Target Discovery Institute, Nuffield Department of Clinical Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK.

Background: Acetylation of lysine residues in histone tails plays an important role in the regulation of gene transcription. Bromdomains are the readers of acetylated histone marks, and, consequently, bromodomain-containing proteins have a variety of chromatin-related functions. Moreover, they are increasingly being recognised as important mediators of a wide range of diseases. The first potent and selective bromodomain inhibitors are beginning to be described, but the diverse or unknown functions of bromodomain-containing proteins present challenges to systematically demonstrating cellular efficacy and selectivity for these inhibitors. Here we assess the viability of fluorescence recovery after photobleaching (FRAP) assays as a target agnostic method for the direct visualisation of an on-target effect of bromodomain inhibitors in living cells.

Results: Mutation of a conserved asparagine crucial for binding to acetylated lysines in the bromodomains of BRD3, BRD4 and TRIM24 all resulted in reduction of FRAP recovery times, indicating loss of or significantly reduced binding to acetylated chromatin, as did the addition of known inhibitors. Significant differences between wild type and bromodomain mutants for ATAD2, BAZ2A, BRD1, BRD7, GCN5L2, SMARCA2 and ZMYND11 required the addition of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) to amplify the binding contribution of the bromodomain. Under these conditions, known inhibitors decreased FRAP recovery times back to mutant control levels. Mutation of the bromodomain did not alter FRAP recovery times for full-length CREBBP, even in the presence of SAHA, indicating that other domains are primarily responsible for anchoring CREBBP to chromatin. However, FRAP assays with multimerised CREBBP bromodomains resulted in a good assay to assess the efficacy of bromodomain inhibitors to this target. The bromodomain and extraterminal protein inhibitor PFI-1 was inactive against other bromodomain targets, demonstrating the specificity of the method.

Conclusions: Viable FRAP assays were established for 11 representative bromodomain-containing proteins that broadly cover the bromodomain phylogenetic tree. Addition of SAHA can overcome weak binding to chromatin, and the use of tandem bromodomain constructs can eliminate masking effects of other chromatin binding domains. Together, these results demonstrate that FRAP assays offer a potentially pan-bromodomain method for generating cell-based assays, allowing the testing of compounds with respect to cell permeability, on-target efficacy and selectivity.
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http://dx.doi.org/10.1186/1756-8935-7-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4115480PMC
August 2014

A scintillation proximity assay for histone demethylases.

Anal Biochem 2014 Oct 8;463:54-60. Epub 2014 Jul 8.

Structural Genomics Consortium, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada. Electronic address:

Covalent modifications, such as methylation and demethylation of lysine residues in histones, play important roles in chromatin dynamics and the regulation of gene expression. The lysine demethylases (KDMs) catalyze the demethylation of lysine residues on histone tails and are associated with diverse human diseases, including cancer, and are therefore proposed as targets for the therapeutic modulation of gene transcription. High-throughput assays have been developed to find inhibitors of KDMs, most of which are fluorescence-based assays. Here we report the development of a coupled scintillation proximity assay (SPA) for 3 KDMs: KDM1A (LSD1), KDM3A (JMJD1A), and KDM4A (JMJD2A). In this assay methylated peptides are first demethylated by a KDM, and a protein methyltransferase (PMT) is added to methylate the resulting peptide with tritiated S-(5'-adenosyl)-l-methionine. The enzyme activities were optimized and kinetic parameters were determined. These robust coupled assays are suitable for screening KDMs in 384-well format (Z' factors of 0.70-0.80), facilitating discovery of inhibitors in the quest for cancer therapeutics.
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http://dx.doi.org/10.1016/j.ab.2014.06.023DOI Listing
October 2014

Medium-throughput production of recombinant human proteins: protein production in insect cells.

Methods Mol Biol 2014 ;1091:95-121

Structural Genomics Consortium, University of Oxford, Oxford, UK.

This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the baculovirus expression vector system (BEVS). This eukaryotic expression system was selected and a screening process established in 2007 as a measure to tackle the more challenging kinase, RNA-DNA processing and integral membrane protein families on our target list. Here, we discuss our platform for identifying soluble proteins from 3 ml of insect cell culture and describe the procedures involved in producing protein from liter-scale cultures. Although not discussed in this chapter, the same process can also be applied to integral membrane proteins (IMPs) with slight adaptations to the purification procedure.
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http://dx.doi.org/10.1007/978-1-62703-691-7_6DOI Listing
June 2014

Medium-throughput production of recombinant human proteins: protein production in E. coli.

Methods Mol Biol 2014 ;1091:73-94

Structural Genomics Consortium, University of Oxford, Oxford, UK.

In Chapter 4 we described the SGC process for generating multiple constructs of truncated versions of each protein using LIC. In this chapter we provide a step-by-step procedure of our E. coli system for test expressing intracellular (soluble) proteins in a 96-well format that enables us to identify which proteins or truncated versions are expressed in a soluble and stable form suitable for structural studies. In addition, we detail the process for scaling up cultures for large-scale protein purification. This level of production is required to obtain sufficient quantities (i.e., milligram amounts) of protein for further characterization and/or crystallization experiments. Our standard process is purification by immobilized metal affinity chromatography (IMAC) using nickel resin followed by size exclusion chromatography (SEC), with additional procedures arising from the complexity of the protein itself.
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http://dx.doi.org/10.1007/978-1-62703-691-7_5DOI Listing
June 2014

Medium-throughput production of recombinant human proteins: ligation-independent cloning.

Methods Mol Biol 2014 ;1091:55-72

Structural Genomics Consortium, University of Oxford, Oxford, UK.

Structural genomics groups have identified the need to generate multiple truncated versions of each target to improve their success in producing a well-expressed, soluble, and stable protein and one that crystallizes and diffracts to a sufficient resolution for structural determination. At the SGC, we opted for the Ligation-Independent Cloning (LIC) method which provides the medium throughput we desire to produce and screen many proteins in a parallel process. Here, we describe our LIC protocol for generating constructs in a 96-well format and provide a choice of vectors suitable for expressing proteins in both E. coli and the baculovirus expression vector system (BEVS).
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http://dx.doi.org/10.1007/978-1-62703-691-7_4DOI Listing
June 2014

Structural insights into functional overlapping and differentiation among myosin V motors.

J Biol Chem 2013 Nov 4;288(47):34131-34145. Epub 2013 Oct 4.

Brazilian Biosciences National Laboratory, National Center for Research in Energy and Materials, Campinas, São Paulo 13083-100, Brazil. Electronic address:

Myosin V (MyoV) motors have been implicated in the intracellular transport of diverse cargoes including vesicles, organelles, RNA-protein complexes, and regulatory proteins. Here, we have solved the cargo-binding domain (CBD) structures of the three human MyoV paralogs (Va, Vb, and Vc), revealing subtle structural changes that drive functional differentiation and a novel redox mechanism controlling the CBD dimerization process, which is unique for the MyoVc subclass. Moreover, the cargo- and motor-binding sites were structurally assigned, indicating the conservation of residues involved in the recognition of adaptors for peroxisome transport and providing high resolution insights into motor domain inhibition by CBD. These results contribute to understanding the structural requirements for cargo transport, autoinhibition, and regulatory mechanisms in myosin V motors.
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http://dx.doi.org/10.1074/jbc.M113.507202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3837155PMC
November 2013

Structures of ABCB10, a human ATP-binding cassette transporter in apo- and nucleotide-bound states.

Proc Natl Acad Sci U S A 2013 Jun 28;110(24):9710-5. Epub 2013 May 28.

Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom.

ABCB10 is one of the three ATP-binding cassette (ABC) transporters found in the inner membrane of mitochondria. In mammals ABCB10 is essential for erythropoiesis, and for protection of mitochondria against oxidative stress. ABCB10 is therefore a potential therapeutic target for diseases in which increased mitochondrial reactive oxygen species production and oxidative stress play a major role. The crystal structure of apo-ABCB10 shows a classic exporter fold ABC transporter structure, in an open-inwards conformation, ready to bind the substrate or nucleotide from the inner mitochondrial matrix or membrane. Unexpectedly, however, ABCB10 adopts an open-inwards conformation when complexed with nonhydrolysable ATP analogs, in contrast to other transporter structures which adopt an open-outwards conformation in complex with ATP. The three complexes of ABCB10/ATP analogs reported here showed varying degrees of opening of the transport substrate binding site, indicating that in this conformation there is some flexibility between the two halves of the protein. These structures suggest that the observed plasticity, together with a portal between two helices in the transmembrane region of ABCB10, assist transport substrate entry into the substrate binding cavity. These structures indicate that ABC transporters may exist in an open-inwards conformation when nucleotide is bound. We discuss ways in which this observation can be aligned with the current views on mechanisms of ABC transporters.
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http://dx.doi.org/10.1073/pnas.1217042110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3683770PMC
June 2013

The structural basis of ZMPSTE24-dependent laminopathies.

Science 2013 Mar;339(6127):1604-7

Structural Genomics Consortium, University of Oxford, Oxford, UK.

Mutations in the nuclear membrane zinc metalloprotease ZMPSTE24 lead to diseases of lamin processing (laminopathies), such as the premature aging disease progeria and metabolic disorders. ZMPSTE24 processes prelamin A, a component of the nuclear lamina intermediate filaments, by cleaving it at two sites. Failure of this processing results in accumulation of farnesylated, membrane-associated prelamin A. The 3.4 angstrom crystal structure of human ZMPSTE24 has a seven transmembrane α-helical barrel structure, surrounding a large, water-filled, intramembrane chamber, capped by a zinc metalloprotease domain with the catalytic site facing into the chamber. The 3.8 angstrom structure of a complex with a CSIM tetrapeptide showed that the mode of binding of the substrate resembles that of an insect metalloprotease inhibitor in thermolysin. Laminopathy-associated mutations predicted to reduce ZMPSTE24 activity map to the zinc metalloprotease peptide-binding site and to the bottom of the chamber.
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http://dx.doi.org/10.1126/science.1231513DOI Listing
March 2013

Plant growth regulator daminozide is a selective inhibitor of human KDM2/7 histone demethylases.

J Med Chem 2012 Jul 11;55(14):6639-43. Epub 2012 Jul 11.

Epigenetic Regulation of Chromatin Function Group, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.

The JmjC oxygenases catalyze the N-demethylation of N(ε)-methyl lysine residues in histones and are current therapeutic targets. A set of human 2-oxoglutarate analogues were screened using a unified assay platform for JmjC demethylases and related oxygenases. Results led to the finding that daminozide (N-(dimethylamino)succinamic acid, 160 Da), a plant growth regulator, selectively inhibits the KDM2/7 JmjC subfamily. Kinetic and crystallographic studies reveal that daminozide chelates the active site metal via its hydrazide carbonyl and dimethylamino groups.
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http://dx.doi.org/10.1021/jm300677jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4673902PMC
July 2012

The PHD and chromo domains regulate the ATPase activity of the human chromatin remodeler CHD4.

J Mol Biol 2012 Sep 7;422(1):3-17. Epub 2012 May 7.

Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK.

The NuRD (nucleosome remodeling and deacetylase) complex serves as a crucial epigenetic regulator of cell differentiation, proliferation, and hematopoietic development by coupling the deacetylation and demethylation of histones, nucleosome mobilization, and the recruitment of transcription factors. The core nucleosome remodeling function of the mammalian NuRD complex is executed by the helicase-domain-containing ATPase CHD4 (Mi-2β) subunit, which also contains N-terminal plant homeodomain (PHD) and chromo domains. The mode of regulation of chromatin remodeling by CHD4 is not well understood, nor is the role of its PHD and chromo domains. Here, we use small-angle X-ray scattering, nucleosome binding ATPase and remodeling assays, limited proteolysis, cross-linking, and tandem mass spectrometry to propose a three-dimensional structural model describing the overall shape and domain interactions of CHD4 and discuss the relevance of these for regulating the remodeling of chromatin by the NuRD complex.
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http://dx.doi.org/10.1016/j.jmb.2012.04.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3437443PMC
September 2012

Expressing the human proteome for affinity proteomics: optimising expression of soluble protein domains and in vivo biotinylation.

N Biotechnol 2012 Jun 19;29(5):515-25. Epub 2011 Oct 19.

The Structural Genomics Consortium, University of Oxford, ORCRB, Roosevelt Drive, Oxford OX3 7DQ, United Kingdom.

The generation of affinity reagents to large numbers of human proteins depends on the ability to express the target proteins as high-quality antigens. The Structural Genomics Consortium (SGC) focuses on the production and structure determination of human proteins. In a 7-year period, the SGC has deposited crystal structures of >800 human protein domains, and has additionally expressed and purified a similar number of protein domains that have not yet been crystallised. The targets include a diversity of protein domains, with an attempt to provide high coverage of protein families. The family approach provides an excellent basis for characterising the selectivity of affinity reagents. We present a summary of the approaches used to generate purified human proteins or protein domains, a test case demonstrating the ability to rapidly generate new proteins, and an optimisation study on the modification of >70 proteins by biotinylation in vivo. These results provide a unique synergy between large-scale structural projects and the recent efforts to produce a wide coverage of affinity reagents to the human proteome.
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http://dx.doi.org/10.1016/j.nbt.2011.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3383991PMC
June 2012

Human SNM1A and XPF-ERCC1 collaborate to initiate DNA interstrand cross-link repair.

Genes Dev 2011 Sep;25(17):1859-70

Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom.

One of the major DNA interstrand cross-link (ICL) repair pathways in mammalian cells is coupled to replication, but the mechanistic roles of the critical factors involved remain largely elusive. Here, we show that purified human SNM1A (hSNM1A), which exhibits a 5'-3' exonuclease activity, can load from a single DNA nick and digest past an ICL on its substrate strand. hSNM1A-depleted cells are ICL-sensitive and accumulate replication-associated DNA double-strand breaks (DSBs), akin to ERCC1-depleted cells. These DSBs are Mus81-induced, indicating that replication fork cleavage by Mus81 results from the failure of the hSNM1A- and XPF-ERCC1-dependent ICL repair pathway. Our results reveal how collaboration between hSNM1A and XPF-ERCC1 is necessary to initiate ICL repair in replicating human cells.
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http://dx.doi.org/10.1101/gad.15699211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3175721PMC
September 2011