Publications by authors named "David C Briggs"

28 Publications

  • Page 1 of 1

A two-site flexible clamp mechanism for RET-GDNF-GFRα1 assembly reveals both conformational adaptation and strict geometric spacing.

Structure 2021 Jul 22;29(7):694-708.e7. Epub 2021 Jan 22.

Signalling and Structural Biology Laboratory, Francis Crick Institute, NW1 1AT London, UK; Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck College, Malet Street, London WC1E 7HX, UK. Electronic address:

RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognize and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here, we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2 and CLD3. Comparison with a cryo-electron microscopy structure of a ligand-engaged zebrafish RET-GDNF-GFRα1a complex indicates conformational changes within a clade-specific CLD3 loop adjacent to the co-receptor. Our observations indicate that RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFRα co-receptors, while its rigid arm recognizes a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualize linear arrays of RET-GDNF-GFRα1a suggesting that a conserved contact stabilizes higher-order species. Our study reveals that ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands.
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http://dx.doi.org/10.1016/j.str.2020.12.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8266384PMC
July 2021

Metabolic precision labeling enables selective probing of O-linked -acetylgalactosamine glycosylation.

Proc Natl Acad Sci U S A 2020 10 28;117(41):25293-25301. Epub 2020 Sep 28.

The Chemical Glycobiology Laboratory, The Francis Crick Institute, NW1 1AT London, United Kingdom;

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe -()-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked -acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched -acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding -acetylglucosamine analog by the epimerase -acetylgalactosamine-4-epimerase (GALE) like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.
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http://dx.doi.org/10.1073/pnas.2007297117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7568240PMC
October 2020

Bioisosteric Discovery of NPA101.3, a Second-Generation RET/VEGFR2 Inhibitor Optimized for Single-Agent Polypharmacology.

J Med Chem 2020 05 28;63(9):4506-4516. Epub 2020 Apr 28.

Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli "Federico II", 80131 Napoli, Italy.

RET receptor tyrosine kinase is a driver oncogene in human cancer. We recently identified the clinical drug candidate Pz-1, which targets RET and VEGFR2. A key metabolite of Pz-1 is its less active demethylated pyrazole analogue. Using bioisosteric substitution methods, here, we report the identification of NPA101.3, lacking the structural liability for demethylation. NPA101.3 showed a selective inhibitory profile and an inhibitory concentration 50 (IC) of <0.003 μM for both RET and VEGFR2. NPA101.3 inhibited phosphorylation of all tested RET oncoproteins as well as VEGFR2 and proliferation of cells transformed by RET. Oral administration of NPA101.3 (10 mg/kg/day) completely prevented formation of tumors induced by RET/C634Y-transformed cells, while it weakened, but did not abrogate, formation of tumors induced by a control oncogene (HRAS/G12V). The balanced synchronous inhibition of both RET and VEGFR2, as well the resistance to demethylation, renders NPA101.3 a potential clinical candidate for RET-driven cancers.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901654PMC
May 2020

Inter-α-inhibitor heavy chain-1 has an integrin-like 3D structure mediating immune regulatory activities and matrix stabilization during ovulation.

J Biol Chem 2020 04 6;295(16):5278-5291. Epub 2020 Mar 6.

Wellcome Centre for Cell-Matrix Research, School of Biological Sciences, Faculty of Biology, Medicine & Health, University of Manchester, Manchester Academic Health Science Centre, Manchester M13 9PT, United Kingdom; Division of Cell-Matrix Biology & Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine & Health, University of Manchester, Manchester Academic Health Science Centre, Manchester M13 9PT, United Kingdom; Lydia Becker Institute of Immunology and Inflammation, Faculty of Biology, Medicine & Health, University of Manchester, Manchester M13 9PL, United Kingdom. Electronic address:

Inter-α-inhibitor is a proteoglycan essential for mammalian reproduction and also plays a less well-characterized role in inflammation. It comprises two homologous "heavy chains" (HC1 and HC2) covalently attached to chondroitin sulfate on the bikunin core protein. Before ovulation, HCs are transferred onto the polysaccharide hyaluronan (HA) to form covalent HC·HA complexes, thereby stabilizing an extracellular matrix around the oocyte required for fertilization. Additionally, such complexes form during inflammatory processes and mediate leukocyte adhesion in the synovial fluids of arthritis patients and protect against sepsis. Here using X-ray crystallography, we show that human HC1 has a structure similar to integrin β-chains, with a von Willebrand factor A domain containing a functional metal ion-dependent adhesion site (MIDAS) and an associated hybrid domain. A comparison of the WT protein and a variant with an impaired MIDAS (but otherwise structurally identical) by small-angle X-ray scattering and analytical ultracentrifugation revealed that HC1 self-associates in a cation-dependent manner, providing a mechanism for HC·HA cross-linking and matrix stabilization. Surprisingly, unlike integrins, HC1 interacted with RGD-containing ligands, such as fibronectin, vitronectin, and the latency-associated peptides of transforming growth factor β, in a MIDAS/cation-independent manner. However, HC1 utilizes its MIDAS motif to bind to and inhibit the cleavage of complement C3, and small-angle X-ray scattering-based modeling indicates that this occurs through the inhibition of the alternative pathway C3 convertase. These findings provide detailed structural and functional insights into HC1 as a regulator of innate immunity and further elucidate the role of HC·HA complexes in inflammation and ovulation.
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http://dx.doi.org/10.1074/jbc.RA119.011916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7170535PMC
April 2020

Cryo-EM structures of the XPF-ERCC1 endonuclease reveal how DNA-junction engagement disrupts an auto-inhibited conformation.

Nat Commun 2020 02 28;11(1):1120. Epub 2020 Feb 28.

Signalling and Structural Biology Laboratory, Francis Crick Institute, NW1 1AT, London, UK.

The structure-specific endonuclease XPF-ERCC1 participates in multiple DNA damage repair pathways including nucleotide excision repair (NER) and inter-strand crosslink repair (ICLR). How XPF-ERCC1 is catalytically activated by DNA junction substrates is not currently understood. Here we report cryo-electron microscopy structures of both DNA-free and DNA-bound human XPF-ERCC1. DNA-free XPF-ERCC1 adopts an auto-inhibited conformation in which the XPF helical domain masks the ERCC1 (HhH) domain and restricts access to the XPF catalytic site. DNA junction engagement releases the ERCC1 (HhH) domain to couple with the XPF-ERCC1 nuclease/nuclease-like domains. Structure-function data indicate xeroderma pigmentosum patient mutations frequently compromise the structural integrity of XPF-ERCC1. Fanconi anaemia patient mutations in XPF often display substantial in-vitro activity but are resistant to activation by ICLR recruitment factor SLX4. Our data provide insights into XPF-ERCC1 architecture and catalytic activation.
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http://dx.doi.org/10.1038/s41467-020-14856-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048804PMC
February 2020

Structural Basis for the Initiation of Glycosaminoglycan Biosynthesis by Human Xylosyltransferase 1.

Structure 2018 06 19;26(6):801-809.e3. Epub 2018 Apr 19.

Department of Life Sciences, Imperial College London, London SW7 2AZ, UK. Electronic address:

Proteoglycans (PGs) are essential components of the animal extracellular matrix and are required for cell adhesion, migration, signaling, and immune function. PGs are composed of a core protein and long glycosaminoglycan (GAG) chains, which often specify PG function. GAG biosynthesis is initiated by peptide O-xylosyltransferases, which transfer xylose onto selected serine residues in the core proteins. We have determined crystal structures of human xylosyltransferase 1 (XT1) in complex with the sugar donor, UDP-xylose, and various acceptor peptides. The structures reveal unique active-site features that, in conjunction with functional experiments, explain the substrate specificity of XT1. A constriction within the peptide binding cleft requires the acceptor serine to be followed by glycine or alanine. The remainder of the cleft can accommodate a wide variety of sequences, but with a general preference for acidic residues. These findings provide a framework for understanding the selectivity of GAG attachment.
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http://dx.doi.org/10.1016/j.str.2018.03.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992326PMC
June 2018

Homodimerization of the Lymph Vessel Endothelial Receptor LYVE-1 through a Redox-labile Disulfide Is Critical for Hyaluronan Binding in Lymphatic Endothelium.

J Biol Chem 2016 Nov 12;291(48):25004-25018. Epub 2016 Oct 12.

From the Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom,

The lymphatic vessel endothelial receptor LYVE-1 is implicated in the uptake of hyaluronan (HA) and trafficking of leukocytes to draining lymph nodes. Yet LYVE-1 has only weak affinity for hyaluronan and depends on receptor clustering and higher order ligand organization for durable binding in lymphatic endothelium. An unusual feature of LYVE-1 not found in other HA receptors is the potential to form disulfide-linked homodimers. However, their influence on function has not been investigated. Here we show LYVE-1 homodimers are the predominant configuration in lymphatic endothelium in vitro and in vivo, and formation solely requires the unpaired cysteine residue Cys-201 within the membrane-proximal domain, yielding a 15-fold higher HA binding affinity and an ∼67-fold slower off-rate than the monomer. Moreover, we show non-dimerizing LYVE-1 mutants fail to bind HA even when expressed at high densities in lymphatic endothelial cells or artificially cross-linked with antibody. Consistent with these findings, small angle X-ray scattering (SAXS) indicates the Cys-201 interchain disulfide forms a hinge that maintains the homodimer in an "open scissors" conformation, likely allowing arrangement of the two HA binding domains for mutual engagement with ligand. Finally, we demonstrate the Cys-201 interchain disulfide is highly labile, and selective reduction with TCEP-HCl disrupts LYVE-1 homodimers, ablating HA binding. These findings reveal binding is dependent not just on clustering but also on the biochemical properties of LYVE-1 homodimers. They also mark LYVE-1 as the first Link protein superfamily member requiring covalent homodimerization for function and suggest the interchain disulfide acts as a redox switch in vivo.
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http://dx.doi.org/10.1074/jbc.M116.736926DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5122770PMC
November 2016

Structural basis of laminin binding to the LARGE glycans on dystroglycan.

Nat Chem Biol 2016 10 15;12(10):810-4. Epub 2016 Aug 15.

Howard Hughes Medical Institute, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa, USA.

Dystroglycan is a highly glycosylated extracellular matrix receptor with essential functions in skeletal muscle and the nervous system. Reduced matrix binding by α-dystroglycan (α-DG) due to perturbed glycosylation is a pathological feature of several forms of muscular dystrophy. Like-acetylglucosaminyltransferase (LARGE) synthesizes the matrix-binding heteropolysaccharide [-glucuronic acid-β1,3-xylose-α1,3-]n. Using a dual exoglycosidase digestion, we confirm that this polysaccharide is present on native α-DG from skeletal muscle. The atomic details of matrix binding were revealed by a high-resolution crystal structure of laminin-G-like (LG) domains 4 and 5 (LG4 and LG5) of laminin-α2 bound to a LARGE-synthesized oligosaccharide. A single glucuronic acid-β1,3-xylose disaccharide repeat straddles a Ca(2+) ion in the LG4 domain, with oxygen atoms from both sugars replacing Ca(2+)-bound water molecules. The chelating binding mode accounts for the high affinity of this protein-carbohydrate interaction. These results reveal a previously uncharacterized mechanism of carbohydrate recognition and provide a structural framework for elucidating the mechanisms underlying muscular dystrophy.
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http://dx.doi.org/10.1038/nchembio.2146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5030134PMC
October 2016

Crystallographic analysis of the laminin β2 short arm reveals how the LF domain is inserted into a regular array of LE domains.

Matrix Biol 2017 01 16;57-58:204-212. Epub 2016 Jul 16.

Department of Life Sciences, Imperial College London, UK. Electronic address:

Laminins are a major constituent of all basement membranes. The polymerisation of laminins at the cell surface is mediated by the three short arms of the cross-shaped laminin heterotrimer. The short arms contain repeats of laminin-type epidermal growth factor-like (LE) domains, interspersed with globular domains of unknown function. A single LF domain is inserted between LE5 and LE6 of the laminin β1 and β2 chains. We report the crystal structure at 1.85Å resolution of the laminin β2 LE5-LF-LE6 region. The LF domain consists of a β-sandwich related to bacterial family 35 carbohydrate binding modules, and more distantly to the L4 domains present in the short arms of laminin α and γ chains. An α-helical region mediates the extensive interaction of the LF domain with LE5. The relative arrangement of LE5 and LE6 is very similar to that of consecutive LE domains in uninterrupted LE tandems. Fitting atomic models to a low-resolution structure of the first eight domains of the laminin β1 chain determined by small-angle X-ray scattering suggests a deviation from the regular LE array at the LE4-LE5 junction. These results advance our understanding of laminin structure.
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http://dx.doi.org/10.1016/j.matbio.2016.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5338690PMC
January 2017

Insights into Collagen Uptake by C-type Mannose Receptors from the Crystal Structure of Endo180 Domains 1-4.

Structure 2015 Nov 15;23(11):2133-42. Epub 2015 Oct 15.

Department of Life Sciences, Imperial College London, London SW7 2AZ, UK. Electronic address:

The C-type mannose receptor and its homolog Endo180 (or uPARAP, for urokinase plasminogen activator receptor-associated protein) mediate the endocytic uptake of collagen by macrophages and fibroblasts. This process is required for normal tissue remodeling, but also facilitates the growth and dissemination of tumors. We have determined the crystal structure at 2.5 Å resolution of the N-terminal region of Endo180, consisting of a ricin-like domain, a fibronectin type II (FN2) domain, and two C-type lectin (CTL) domains. The L-shaped arrangement of these domains creates a shallow trench spanning the FN2 and CTL1 domains, which was shown by mutagenesis to bind triple-helical and denatured collagen. Small-angle X-ray scattering showed that the L-shaped structure is maintained in solution at neutral and acidic pH, irrespective of calcium ion loading. Collagen binding was equally unaffected by acidic pH, suggesting that collagen release in endosomes is not regulated by changes within the Endo180 N-terminal region.
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http://dx.doi.org/10.1016/j.str.2015.09.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4635314PMC
November 2015

Metal Ion-dependent Heavy Chain Transfer Activity of TSG-6 Mediates Assembly of the Cumulus-Oocyte Matrix.

J Biol Chem 2015 Nov 14;290(48):28708-23. Epub 2015 Oct 14.

From the Wellcome Trust Centre for Cell-Matrix Research and the Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom,

The matrix polysaccharide hyaluronan (HA) has a critical role in the expansion of the cumulus cell-oocyte complex (COC), a process that is necessary for ovulation and fertilization in most mammals. Hyaluronan is organized into a cross-linked network by the cooperative action of three proteins, inter-α-inhibitor (IαI), pentraxin-3, and TNF-stimulated gene-6 (TSG-6), driving the expansion of the COC and providing the cumulus matrix with its required viscoelastic properties. Although it is known that matrix stabilization involves the TSG-6-mediated transfer of IαI heavy chains (HCs) onto hyaluronan (to form covalent HC·HA complexes that are cross-linked by pentraxin-3) and that this occurs via the formation of covalent HC·TSG-6 intermediates, the underlying molecular mechanisms are not well understood. Here, we have determined the tertiary structure of the CUB module from human TSG-6, identifying a calcium ion-binding site and chelating glutamic acid residue that mediate the formation of HC·TSG-6. This occurs via an initial metal ion-dependent, non-covalent, interaction between TSG-6 and HCs that also requires the presence of an HC-associated magnesium ion. In addition, we have found that the well characterized hyaluronan-binding site in the TSG-6 Link module is not used for recognition during transfer of HCs onto HA. Analysis of TSG-6 mutants (with impaired transferase and/or hyaluronan-binding functions) revealed that although the TSG-6-mediated formation of HC·HA complexes is essential for the expansion of mouse COCs in vitro, the hyaluronan-binding function of TSG-6 does not play a major role in the stabilization of the murine cumulus matrix.
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http://dx.doi.org/10.1074/jbc.M115.669838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4661386PMC
November 2015

Incorporation of pentraxin 3 into hyaluronan matrices is tightly regulated and promotes matrix cross-linking.

J Biol Chem 2014 Oct 4;289(44):30481-30498. Epub 2014 Sep 4.

CIC biomaGUNE, 20009 Donostia-San Sebastian, Spain,; Department of Molecular Chemistry, University Grenoble Alpes and CNRS, 38000 Grenoble, France, and; Max-Planck-Institute for Intelligent Systems, 70569 Stuttgart, Germany. Electronic address:

Mammalian oocytes are surrounded by a highly hydrated hyaluronan (HA)-rich extracellular matrix with embedded cumulus cells, forming the cumulus cell·oocyte complex (COC) matrix. The correct assembly, stability, and mechanical properties of this matrix, which are crucial for successful ovulation, transport of the COC to the oviduct, and its fertilization, depend on the interaction between HA and specific HA-organizing proteins. Although the proteins inter-α-inhibitor (IαI), pentraxin 3 (PTX3), and TNF-stimulated gene-6 (TSG-6) have been identified as being critical for COC matrix formation, its supramolecular organization and the molecular mechanism of COC matrix stabilization remain unknown. Here we used films of end-grafted HA as a model system to investigate the molecular interactions involved in the formation and stabilization of HA matrices containing TSG-6, IαI, and PTX3. We found that PTX3 binds neither to HA alone nor to HA films containing TSG-6. This long pentraxin also failed to bind to products of the interaction between IαI, TSG-6, and HA, among which are the covalent heavy chain (HC)·HA and HC·TSG-6 complexes, despite the fact that both IαI and TSG-6 are ligands of PTX3. Interestingly, prior encounter with IαI was required for effective incorporation of PTX3 into TSG-6-loaded HA films. Moreover, we demonstrated that this ternary protein mixture made of IαI, PTX3, and TSG-6 is sufficient to promote formation of a stable (i.e. cross-linked) yet highly hydrated HA matrix. We propose that this mechanism is essential for correct assembly of the COC matrix and may also have general implications in other inflammatory processes that are associated with HA cross-linking.
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http://dx.doi.org/10.1074/jbc.M114.568154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4215230PMC
October 2014

A refined model for the TSG-6 link module in complex with hyaluronan: use of defined oligosaccharides to probe structure and function.

J Biol Chem 2014 Feb 8;289(9):5619-34. Epub 2014 Jan 8.

From the Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.

Tumor necrosis factor-stimulated gene-6 (TSG-6) is an inflammation-associated hyaluronan (HA)-binding protein that contributes to remodeling of HA-rich extracellular matrices during inflammatory processes and ovulation. The HA-binding domain of TSG-6 consists solely of a Link module, making it a prototypical member of the superfamily of proteins that interacts with this high molecular weight polysaccharide composed of repeating disaccharides of D-glucuronic acid and N-acetyl-D-glucosamine (GlcNAc). Previously we modeled a complex of the TSG-6 Link module in association with an HA octasaccharide based on the structure of the domain in its HA-bound conformation. Here we have generated a refined model for a HA/Link module complex using novel restraints identified from NMR spectroscopy of the protein in the presence of 10 distinct HA oligosaccharides (from 4- to 8-mers); the model was then tested using unique sugar reagents, i.e. chondroitin/HA hybrid oligomers and an octasaccharide in which a single sugar ring was (13)C-labeled. The HA chain was found to make more extensive contacts with the TSG-6 surface than thought previously, such that a D-glucuronic acid ring makes stacking and ionic interactions with a histidine and lysine, respectively. Importantly, this causes the HA to bend around two faces of the Link module (resembling the way that HA binds to CD44), potentially providing a mechanism for how TSG-6 can reorganize HA during inflammation. However, the HA-binding site defined here may not play a role in TSG-6-mediated transfer of heavy chains from inter-α-inhibitor onto HA, a process known to be essential for ovulation.
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http://dx.doi.org/10.1074/jbc.M113.542357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3937638PMC
February 2014

Inter-α-inhibitor impairs TSG-6-induced hyaluronan cross-linking.

J Biol Chem 2013 Oct 4;288(41):29642-53. Epub 2013 Sep 4.

From the Biosurfaces Unit, CIC biomaGUNE, 20009 Donostia-San Sebastian, Spain.

Under inflammatory conditions and in the matrix of the cumulus-oocyte complex, the polysaccharide hyaluronan (HA) becomes decorated covalently with heavy chains (HCs) of the serum glycoprotein inter-α-inhibitor (IαI). This alters the functional properties of the HA as well as its structural role within extracellular matrices. The covalent transfer of HCs from IαI to HA is catalyzed by TSG-6 (tumor necrosis factor-stimulated gene-6), but TSG-6 is also known as a HA cross-linker that induces condensation of the HA matrix. Here, we investigate the interplay of these two distinct functions of TSG-6 by studying the ternary interactions of IαI and TSG-6 with well defined films of end-grafted HA chains. We demonstrate that TSG-6-mediated cross-linking of HA films is impaired in the presence of IαI and that this effect suppresses the TSG-6-mediated enhancement of HA binding to CD44-positive cells. Furthermore, we find that the interaction of TSG-6 and IαI in the presence of HA gives rise to two types of complexes that independently promote the covalent transfer of heavy chains to HA. One type of complex interacts very weakly with HA and is likely to correspond to the previously reported covalent HC·TSG-6 complexes. The other type of complex is novel and binds stably but noncovalently to HA. Prolonged incubation with TSG-6 and IαI leads to HA films that contain, in addition to covalently HA-bound HCs, several tightly but noncovalently bound molecular species. These findings have important implications for understanding how the biological activities of TSG-6 are regulated, such that the presence or absence of IαI will dictate its function.
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http://dx.doi.org/10.1074/jbc.M113.477422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795262PMC
October 2013

Structure of the food-poisoning Clostridium perfringens enterotoxin reveals similarity to the aerolysin-like pore-forming toxins.

J Mol Biol 2011 Oct 3;413(1):138-49. Epub 2011 Aug 3.

Department of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, Malet Street, London WC1E 7HX, UK.

Clostridium perfringens enterotoxin (CPE) is a major cause of food poisoning and antibiotic-associated diarrhea. Upon its release from C. perfringens spores, CPE binds to its receptor, claudin, at the tight junctions between the epithelial cells of the gut wall and subsequently forms pores in the cell membranes. A number of different complexes between CPE and claudin have been observed, and the process of pore formation has not been fully elucidated. We have determined the three-dimensional structure of the soluble form of CPE in two crystal forms by X-ray crystallography, to a resolution of 2.7 and 4.0 Å, respectively, and found that the N-terminal domain shows structural homology with the aerolysin-like β-pore-forming family of proteins. We show that CPE forms a trimer in both crystal forms and that this trimer is likely to be biologically relevant but is not the active pore form. We use these data to discuss models of pore formation.
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http://dx.doi.org/10.1016/j.jmb.2011.07.066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3235586PMC
October 2011

The inflammation-associated protein TSG-6 cross-links hyaluronan via hyaluronan-induced TSG-6 oligomers.

J Biol Chem 2011 Jul 19;286(29):25675-86. Epub 2011 May 19.

Biosurfaces Unit, CIC biomaGUNE, Paseo Miramon 182, 20009 Donostia-San Sebastian, Spain.

Tumor necrosis factor-stimulated gene-6 (TSG-6) is a hyaluronan (HA)-binding protein that plays important roles in inflammation and ovulation. TSG-6-mediated cross-linking of HA has been proposed as a functional mechanism (e.g. for regulating leukocyte adhesion), but direct evidence for cross-linking is lacking, and we know very little about its impact on HA ultrastructure. Here we used films of polymeric and oligomeric HA chains, end-grafted to a solid support, and a combination of surface-sensitive biophysical techniques to quantify the binding of TSG-6 into HA films and to correlate binding to morphological changes. We find that full-length TSG-6 binds with pronounced positive cooperativity and demonstrate that it can cross-link HA at physiologically relevant concentrations. Our data indicate that cooperative binding of full-length TSG-6 arises from HA-induced protein oligomerization and that the TSG-6 oligomers act as cross-linkers. In contrast, the HA-binding domain of TSG-6 (the Link module) alone binds without positive cooperativity and weaker than the full-length protein. Both the Link module and full-length TSG-6 condensed and rigidified HA films, and the degree of condensation scaled with the affinity between the TSG-6 constructs and HA. We propose that condensation is the result of protein-mediated HA cross-linking. Our findings firmly establish that TSG-6 is a potent HA cross-linking agent and might hence have important implications for the mechanistic understanding of the biological function of TSG-6 (e.g. in inflammation).
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http://dx.doi.org/10.1074/jbc.M111.247395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3138277PMC
July 2011

Molecular recognition of the Tes LIM2-3 domains by the actin-related protein Arp7A.

J Biol Chem 2011 Apr 29;286(13):11543-54. Epub 2011 Jan 29.

Cell Motility Laboratory, Cancer Research UK, London Research Institute, London, United Kingdom.

Actin-related proteins (Arps) are a highly conserved family of proteins that have extensive sequence and structural similarity to actin. All characterized Arps are components of large multimeric complexes associated with chromatin or the cytoskeleton. In addition, the human genome encodes five conserved but largely uncharacterized "orphan" Arps, which appear to be mostly testis-specific. Here we show that Arp7A, which has 43% sequence identity with β-actin, forms a complex with the cytoskeletal proteins Tes and Mena in the subacrosomal layer of round spermatids. The N-terminal 65-residue extension to the actin-like fold of Arp7A interacts directly with Tes. The crystal structure of the 1-65(Arp7A)·LIM2-3(Tes)·EVH1(Mena) complex reveals that residues 28-49 of Arp7A contact the LIM2-3 domains of Tes. Two alanine residues from Arp7A that occupy equivalent apolar pockets in both LIM domains as well as an intervening GPAK linker that binds the LIM2-3 junction are critical for the Arp7A-Tes interaction. Equivalent occupied apolar pockets are also seen in the tandem LIM domain structures of LMO4 and Lhx3 bound to unrelated ligands. Our results indicate that apolar pocket interactions are a common feature of tandem LIM domain interactions, but ligand specificity is principally determined by the linker sequence.
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http://dx.doi.org/10.1074/jbc.M110.171264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3064208PMC
April 2011

Crystallization and preliminary crystallographic analysis of the Clostridium perfringens enterotoxin.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2010 Jul 24;66(Pt 7):794-7. Epub 2010 Jun 24.

Birkbeck College, University of London, England.

Clostridium perfringens is a Gram-positive anaerobic species of bacterium that is notable for its ability to produce a plethora of toxins, including membrane-active toxins (alpha-toxins), pore-forming toxins (-toxins) and binary toxins (iota-toxins). Here, the crystallization of the full-length wild-type C. perfringens enterotoxin is reported, which is the causative agent of the second most prevalent food-borne illness in the United States and has been implicated in many other gastrointestinal pathologies. Several crystal forms were obtained. However, only two of these optimized crystal forms (I and II) were useable for X-ray diffraction data collection. The form I crystals diffracted to d(min) = 2.7 A and belonged to space group C2, while the form II crystals diffracted to d(min) = 4 A and belonged to space group P2(1)3.
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http://dx.doi.org/10.1107/S1744309110016507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2898463PMC
July 2010

The angiogenic inhibitor long pentraxin PTX3 forms an asymmetric octamer with two binding sites for FGF2.

J Biol Chem 2010 Jun 2;285(23):17681-92. Epub 2010 Apr 2.

Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.

The inflammation-associated long pentraxin PTX3 plays key roles in innate immunity, female fertility, and vascular biology (e.g. it inhibits FGF2 (fibroblast growth factor 2)-mediated angiogenesis). PTX3 is composed of multiple protomers, each composed of distinct N- and C-terminal domains; however, it is not known how these are organized or contribute to its functional properties. Here, biophysical analyses reveal that PTX3 is composed of eight identical protomers, associated through disulfide bonds, forming an elongated and asymmetric, molecule with two differently sized domains interconnected by a stalk. The N-terminal region of the protomer provides the main structural determinant underlying this quaternary organization, supporting formation of a disulfide-linked tetramer and a dimer of dimers (a non-covalent tetramer), giving rise to the asymmetry of the molecule. Furthermore, the PTX3 octamer is shown to contain two FGF2 binding sites, where it is the tetramers that act as the functional units in ligand recognition. Thus, these studies provide a unifying model of the PTX3 oligomer, explaining both its quaternary organization and how this is required for its antiangiogenic function.
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http://dx.doi.org/10.1074/jbc.M109.085639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2878532PMC
June 2010

Single nucleotide polymorphism analysis of the NKG2D ligand cluster on the long arm of chromosome 6: Extensive polymorphisms and evidence of diversity between human populations.

Hum Immunol 2010 Jun 15;71(6):610-20. Epub 2010 Mar 15.

School of Cancer Sciences, Birmingham University, Birmingham, United Kingdom.

NKG2D is an important activating receptor on NK cells and T-cells and has a diverse panel of ligands (NKG2DL) which include the ULBP and RAET1 proteins. Several NKG2DL exhibit a considerable degree of genetic polymorphism, and although the functional significance of such allelic variation remains unclear, genetic variants have been implicated in susceptibility to infection and auto-immune disease. We used sequence-specific primer polymerase chain reaction to determine the frequency of 25 single nucleotide polymorphisms (SNPs) in the promoter and coding regions of genes of the RAET1/ULBP cluster in 223 Euro-Caucasoid, 60 Afro-Caribbean, and 52 Indo-Asian individuals to determine NKG2DL allele and haplotype frequencies within these populations. We show marked differences in the frequency of NKG2DL SNPs and haplotypes among the three ethnic groups, and certain haplotypes were observed almost exclusively in Afro-Caribbean compared with the Euro-Caucasoid and Indo-Asian populations. Interestingly, variation was focused within the RAET1E (ULBP4), RAET1L, and ULBP3 genes, whereas the ULBP1, ULBP2 and RAET1G (ULBP5) genes were highly conserved. These findings suggest that individual NKG2DL alleles have been subject to divergent selective pressures during the migration of Homo sapiens. This information will be of importance in understanding the biology and clinical significance of NKG2DL polymorphism.
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http://dx.doi.org/10.1016/j.humimm.2010.02.018DOI Listing
June 2010

Association of killer cell immunoglobulin-like receptors with primary Sjogren's syndrome.

Rheumatology (Oxford) 2009 Apr 30;48(4):359-62. Epub 2009 Jan 30.

Department of Histocompatibility and Immunogenetics, NHS Blood and Transplant, Vincent Drive, Edgbaston, Birmingham, UK.

Objective: SS is a chronic inflammatory condition characterized by systemic and tissue-specific autoimmune features. In view of recent findings indicating a role for killer cell immunoglobulin-like receptors (KIRs) in the pathogenesis of other autoimmune rheumatic disorders such as SSc, and the autoimmune disorders RA and PsA, we sought to determine whether KIRs predict general or specific susceptibility in SS.

Methods: Eleven separate KIR genes were typed using PCR sequence-specific primers on genomic DNA from 72 patients diagnosed with primary SS and a control panel consisting of 223 blood donors.

Results: We found no individual KIR genes to be associated with SS. In contrast, 11 patients with primary SS (15%) and 9 control blood donors (4%) had KIR genotypes with the activating KIR2DS2 in the absence of its corresponding inhibitory homologue KIR2DL2 (P = 0.01). Further analysis of these individuals showed that seven SS patients were positive for HLA-C ligand for KIR2DS2 only compared with one control sample (P = 0.00026).

Conclusion: The genetic combination of KIR2DS2+ and KIR2DL2- in the presence of HLA-C ligand specific for activating KIR2DS2 is associated with primary SS. This implies that autologous KIR-ligand interaction is a contributory factor to predisposition for this disease.
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http://dx.doi.org/10.1093/rheumatology/ken503DOI Listing
April 2009

A bug in CUB's clothing: similarity between clostridial CBMs and complement CUBs.

Trends Microbiol 2008 Sep 6;16(9):407-8. Epub 2008 Aug 6.

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http://dx.doi.org/10.1016/j.tim.2008.06.002DOI Listing
September 2008

Tes, a specific Mena interacting partner, breaks the rules for EVH1 binding.

Mol Cell 2007 Dec;28(6):1071-82

Cell Motility Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK.

The intracellular targeting of Ena/VASP family members is achieved via the interaction of their EVH1 domain with FPPPP sequence motifs found in a variety of cytoskeletal proteins, including lamellipodin, vinculin, and zyxin. Here we show that the LIM3 domain of Tes, which lacks the FPPPP motif, binds to the EVH1 domain of Mena, but not to those of VASP or Evl. The structure of the LIM3:EVH1 complex reveals that Tes occludes the FPPPP-binding site and competes with FPPPP-containing proteins for EVH1 binding. Structure-based gain-of-function experiments define the molecular basis for the specificity of the Tes-Mena interaction. Consistent with in vitro observations, the LIM3 domain displaces Mena, but not VASP, from the leading edge and focal adhesions. It also regulates cell migration through a Mena-dependent mechanism. Our observations identify Tes as an atypical EVH1 binding partner and a regulator specific to a single Ena/VASP family member.
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http://dx.doi.org/10.1016/j.molcel.2007.10.033DOI Listing
December 2007

Structural relationships and cellular tropism of staphylococcal superantigen-like proteins.

Infect Immun 2004 Jul;72(7):4261-70

Department of Immunology and Molecular Pathology, University College London, 46 Cleveland Street, London W1T 4JF, United Kingdom.

The staphylococcal superantigen-like proteins (SSLs) are a family of polymorphic paralogs encoded in the Staphylococcus aureus genome whose function is unknown. The crystal structure of SSL7 was determined and compared to that of SSL5 and that of a classical superantigen, streptococcal pyrogenic exotoxin. Although the overall architecture of the superantigen family is retained in both SSL7 and SSL5, there are significant differences in the structures which suggest that the characteristic major histocompatibility complex binding site of superantigens has been lost. To complement these data, the abilities of SSL7 and a closely related paralog, SSL9, to interact with cells of the immune system were investigated. In populations of human white blood cells, both SSLs interacted selectively with monocytes via specific saturable but separate binding sites, which led to rapid uptake of the SSLs. In addition, SSLs were rapidly taken up by dendritic cells, but not by macrophages, into the same endosomal compartment as dextran. The ability of these secreted proteins to target antigen-presenting cells may enhance a misplaced antibody response against the proteins, which may facilitate bacterial colonization rather than contribute to host protection. Like classical superantigens, therefore, SSLs may distract the host's immune system, but they may do so via entirely different molecular mechanisms.
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http://dx.doi.org/10.1128/IAI.72.7.4261-4270.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC427445PMC
July 2004

Clostridium absonum alpha-toxin: new insights into clostridial phospholipase C substrate binding and specificity.

J Mol Biol 2003 Oct;333(4):759-69

School of Crystallography, Birkbeck College, Malet Street, London WC1E 7HX, UK.

Clostridium absonum phospholipase C (Caa) is a 42.7 kDa protein, which shows 60% amino acid sequence identity with the Clostridium perfringens phospholipase C, or alpha-toxin (Cpa), and has been isolated from patients suffering from gas gangrene. We report the cloning and sequencing, purification, characterisation and crystal structure of the Caa enzyme. Caa had twice the phospholipid-hydrolysing (lecithinase) activity, 1.5 times the haemolytic activity and over seven times the activity towards phosphatidylcholine-based liposomes when compared with Cpa. However, the Caa enzyme had a lower activity than Cpa to the free (i.e. not in lipid bilayer) substrate para-nitrophenylphosphorylcholine, towards sphingomyelin-based liposomes and showed half the cytotoxicity. The lethal dose (LD(50)) of Caa in mice was approximately twice that of Cpa. The crystal structure of Caa shows that the 72-93 residue loop is in a conformation different from those of previously determined open-form alpha-toxin structures. This conformational change suggests a role for W84 in membrane binding and a possible route of entry into the active site along a hydrophobic channel created by the re-arrangement of this loop. Overall, the properties of Caa are compatible with a role as a virulence-determinant in gas gangrene caused by C.absonum.
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http://dx.doi.org/10.1016/j.jmb.2003.07.016DOI Listing
October 2003

The impact of donor KIR and patient HLA-C genotypes on outcome following HLA-identical sibling hematopoietic stem cell transplantation for myeloid leukemia.

Blood 2004 Feb 22;103(4):1521-6. Epub 2003 Sep 22.

Cancer Research United Kingdom, Institute for Cancer Studies, University of Birmingham.

Killer immunoglobulin-like receptors (KIRs) regulate cell activity of natural killer (NK) cells and some T cells. The predominant ligand for inhibitory KIRs is HLA-C, which subdivides into 2 groups based on the specificity of inhibitory KIRs. The ligands for activatory KIRs are unknown. Following hematopoietic stem cell transplantation (HSCT), recipient tissues may not express a ligand for KIRs present within the graft, and the combination of donor KIR and recipient HLA-C types could influence outcome. HLA and KIR genotypes were determined in 220 donor-recipient pairs from HLA-matched sibling HSCTs performed for myeloid (n = 112) and lymphoid (n = 108) diseases. In HSCTs performed for myeloid disease, overall survival was worse in patients homozygous for group 2 HLA-C (C2) than in patients who carried a group 1 HLA-C (C1) allele (P <.005). Moreover, this effect is seen only when the donor additionally carries the activating KIR gene KIR2DS2 (P =.045). No effect was seen in patients with lymphoid disease. Thus, in HLA-matched sibling HSCT for myeloid leukemia, patients homozygous for C2 alleles receiving a graft from a donor carrying the KIR gene KIR2DS2 have a significantly reduced chance of survival.
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http://dx.doi.org/10.1182/blood-2003-02-0438DOI Listing
February 2004

A multi-laboratory characterization of the KIR genotypes of 10th International Histocompatibility Workshop cell lines.

Hum Immunol 2003 May;64(5):567-71

Cancer Research, UK Institute for Cancer Studies, University of Birmingham, Birmingham, United Kingdom.

Killer immunoglobulinlike receptors (KIRs) are expressed on natural killer and T cells. Both inhibitory and noninhibitory forms have been described, leading to inhibition or continuation of cellular killing activity. The natural ligands identified so far of KIRs are class I human leukocyte antigens (HLA). In particular, the interaction of some KIRs with HLA-Cw has been well characterized. Recent work has implicated KIRs in affecting the outcome of hematopoietic stem-cell transplant (HSCT). This may well lead to a requirement for prospective KIR typing of donor and recipient. We have utilized different typing systems (two using polymerase chain reaction-sequence-specific primers, and one using polymerase chain reaction-sequence-specific oligonucleotide probes) in three separate laboratories to characterize the KIR gene complement of 25 cell lines from the 10th International Histocompatibility Workshop. There were consistent results in 22, and minor differences in 3. When compared with previous results for some of these cell lines, no further differences were found. The differences are due to typing of KIRs KIR2DL1 and KIR2DS5, and may be explained by technical differences or the inability to type new variants. Further improvements in typing may be required if population and clinical studies are to produce accurate results.
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http://dx.doi.org/10.1016/s0198-8859(03)00042-9DOI Listing
May 2003
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