I24-Microfocus Macromolecular Crystallography
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Ilaria
Gori
,
Roger
George
,
Andrew G.
Purkiss
,
Stephanie
Strohbuecker
,
Rebecca A
Randall
,
Roksana
Ogrodowicz
,
Virginie
Carmignac
,
Laurence
Faivre
,
Dhira
Joshi
,
Svend
Kjær
,
Caroline S
Hill
Diamond Proposal Number(s):
[13775]
Open Access
Abstract: Shprintzen–Goldberg syndrome (SGS) is a multisystemic connective tissue disorder, with considerable clinical overlap with Marfan and Loeys–Dietz syndromes. These syndromes have commonly been associated with enhanced TGF-β signaling. In SGS patients, heterozygous point mutations have been mapped to the transcriptional co-repressor SKI, which is a negative regulator of TGF-β signaling that is rapidly degraded upon ligand stimulation. The molecular consequences of these mutations, however, are not understood. Here we use a combination of structural biology, genome editing, and biochemistry to show that SGS mutations in SKI abolish its binding to phosphorylated SMAD2 and SMAD3. This results in stabilization of SKI and consequently attenuation of TGF-β responses, both in knockin cells expressing an SGS mutation and in fibroblasts from SGS patients. Thus, we reveal that SGS is associated with an attenuation of TGF-β-induced transcriptional responses, and not enhancement, which has important implications for other Marfan-related syndromes.
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Jan 2021
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13775]
Open Access
Abstract: IGF2 mRNA-binding protein 1 (IMP1) is a key regulator of messenger RNA (mRNA) metabolism and transport in organismal development and, in cancer, its mis-regulation is an important component of tumour metastasis. IMP1 function relies on the recognition of a diverse set of mRNA targets that is mediated by the combinatorial action of multiple RNA-binding domains. Here, we dissect the structure and RNA-binding properties of two key RNA-binding domains of IMP1, KH1 and KH2, and we build a kinetic model for the recognition of RNA targets. Our data and model explain how the two domains are organized as an intermolecular pseudo-dimer and that the important role they play in mRNA target recognition is underpinned by the high RNA-binding affinity and fast kinetics of this KH1KH2–RNA recognition unit. Importantly, the high-affinity RNA-binding by KH1KH2 is achieved by an inter-domain coupling 50-fold stronger than that existing in a second pseudo-dimer in the protein, KH3KH4. The presence of this strong coupling supports a role of RNA re-modelling in IMP1 recognition of known cancer targets.
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Mar 2019
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I03-Macromolecular Crystallography
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Fruzsina
Hobor
,
Andre
Dallmann
,
Neil J.
Ball
,
Carla
Cicchini
,
Cecilia
Battistelli
,
Roksana W.
Ogrodowicz
,
Evangelos
Christodoulou
,
Stephen R.
Martin
,
Alfredo
Castello
,
Marco
Tripodi
,
Ian A.
Taylor
,
Andres
Ramos
Diamond Proposal Number(s):
[13775]
Open Access
Abstract: Exosomal miRNA transfer is a mechanism for cell–cell communication that is important in the immune response, in the functioning of the nervous system and in cancer. Syncrip/hnRNPQ is a highly conserved RNA-binding protein that mediates the exosomal partition of a set of miRNAs. Here, we report that Syncrip’s amino-terminal domain, which was previously thought to mediate protein–protein interactions, is a cryptic, conserved and sequence-specific RNA-binding domain, designated NURR (N-terminal unit for RNA recognition). The NURR domain mediates the specific recognition of a short hEXO sequence defining Syncrip exosomal miRNA targets, and is coupled by a non-canonical structural element to Syncrip’s RRM domains to achieve high-affinity miRNA binding. As a consequence, Syncrip-mediated selection of the target miRNAs implies both recognition of the hEXO sequence by the NURR domain and binding of the RRM domains 5′ to this sequence. This structural arrangement enables Syncrip-mediated selection of miRNAs with different seed sequences.
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Feb 2018
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7707, 9826]
Open Access
Abstract: ATP-phosphoribosyltransferase (ATP-PRT) is a hexameric enzyme in conformational equilibrium between an open and seemingly active state and a closed and presumably inhibited form. The structure-function relationship of allosteric regulation in this system is still not fully understood. Here, we develop a screening strategy for modulators of ATP-PRT and identify 3-(2-thienyl)-l-alanine (TIH) as an allosteric activator of this enzyme. Kinetic analysis reveals co-occupancy of the allosteric sites by TIH and l-histidine. Crystallographic and native ion-mobility mass spectrometry data show that the TIH-bound activated form of the enzyme closely resembles the inhibited l-histidine-bound closed conformation, revealing the uncoupling between ATP-PRT open and closed conformations and its functional state. These findings suggest that dynamic processes are responsible for ATP-PRT allosteric regulation and that similar mechanisms might also be found in other enzymes bearing a ferredoxin-like allosteric domain.
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Aug 2017
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I04-Macromolecular Crystallography
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Abstract: Influenza A haemagglutinin is a surface glycoprotein of Influenza virus, responsible for the initial attachment of the virus to the target cell and, at a later stage, for viral membrane fusion. At the acidic pH of the endosome, the HA molecule undergoes an irreversible structural rearrangement. In consequence, the hydrophobic terminal segments of HA2 are moved to the same end of the refolded molecule, promoting membrane fusion. 16 haemagglutinin subtypes (H1-H16) identified to date can be divided into two groups based on characteristic structural features. The low pH-induced structures of proteolytically prepared and E.coli-expressed fragments of influenza A H3 HA2 (group 2 HA) were previously determined by X-ray crystallography. This study presents structures of proteolytically prepared and recombinantly- expressed fragments of H1 HA2 in a postfusion conformation. Refolded H1 HA2, belonging to group 1 HA, adopts a hairpin-like conformation, similar to that of a rearranged H3 HA2. Structures were compared to the known structures of low pH-activated HA2, to gain a better understanding of the structural differences between the two groups of HA. The data show the structures of the refolded HA2 to be conserved between the HA groups with minor differences. These structural data are supplemented with functional studies involving the cross-reactive FI6 antibody. FI6 antibody binds near the conserved fusion subdomain of the HA molecule and thus interferes with the low pH-triggered conformational change of HA. Additional methods employed in this study, such as limited proteolysis, electron microscopy, biolayer interferometry and MDCK1 cell infection, give insight into the mechanism of FI6 antibody-mediated neutralization, and highlight the differences in infectivity of H1N1 and H3N2 viruses neutralized by the FI6 antibody.
Studies on low pH-activated HA2 from Influenza haemagglutinin | Request PDF. Available from: https://www.researchgate.net/publication/316095388_Studies_on_low_pH-activated_HA2_from_Influenza_haemagglutinin [accessed Jan 23 2018].
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Feb 2017
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Nicole l.
Kallewaard
,
Davide
Corti
,
Patrick J.
Collins
,
Ursula
Neu
,
Josephine M.
Mcauliffe
,
Ebony
Benjamin
,
Leslie
Wachter-Rosati
,
Frances J.
Palmer-Hill
,
Andy Q.
Yuan
,
Philip A.
Walker
,
Matthias
Vorleander
,
Siro
Bianchi
,
Barbara
Guarino
,
Anna
De marco
,
Fabrizia
Vanzetta
,
Gloria
Agatic
,
Mathilde
Foglierini
,
Debora
Pinna
,
Blanca
Fernandez-Rodriguez
,
Alexander
Fruehwirth
,
Chiara
Silacci
,
Roksana W.
Ogrodowicz
,
Stephen R.
Martin
,
Federica
Sallusto
,
Joann A.
Suzich
,
Antonio
Lanzavecchia
,
Qing
Zhu
,
Steven J.
Gamblin
,
John J.
Skehel
Diamond Proposal Number(s):
[9826]
Open Access
Abstract: Influenza virus remains a threat because of its ability to evade vaccine-induced immune responses due to antigenic drift. Here, we describe the isolation, evolution, and structure of a broad-spectrum human monoclonal antibody (mAb), MEDI8852, effectively reacting with all influenza A hemagglutinin (HA) subtypes. MEDI8852 uses the heavy-chain VH6-1 gene and has higher potency and breadth when compared to other anti-stem antibodies. MEDI8852 is effective in mice and ferrets with a therapeutic window superior to that of oseltamivir. Crystallographic analysis of Fab alone or in complex with H5 or H7 HA proteins reveals that MEDI8852 binds through a coordinated movement of CDRs to a highly conserved epitope encompassing a hydrophobic groove in the fusion domain and a large portion of the fusion peptide, distinguishing it from other structurally characterized cross-reactive antibodies. The unprecedented breadth and potency of neutralization by MEDI8852 support its development as immunotherapy for influenza virus-infected humans.
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Jul 2016
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7707]
Abstract: In 2004 an hemagglutinin 3 neuraminidase 8 (H3N8) equine influenza virus was transmitted from horses to dogs in Florida and subsequently spread throughout the United States and to Europe. To understand the molecular basis of changes in the antigenicity of H3 hemagglutinins (HAs) that have occurred during virus evolution in horses, and to investigate the role of HA in the equine to canine cross-species transfer, we used X-ray crystallography to determine the structures of the HAs from two antigenically distinct equine viruses and from a canine virus. Structurally all three are very similar with the majority of amino acid sequence differences between the two equine HAs located on the virus membrane-distal molecular surface. HAs of canine viruses are distinct in containing a Trp-222→Leu substitution in the receptor binding site that influences specificity for receptor analogs. In the fusion subdomain of canine and recent equine virus HAs a unique difference is observed by comparison with all other HAs examined to date. Analyses of site-specific mutant HAs indicate that a single amino acid substitution, Thr-30→Ser, influences interactions between N-terminal and C-terminal regions of the subdomain that are important in the structural changes required for membrane fusion activity. Both structural modifications may have facilitated the transmission of H3N8 influenza from horses to dogs.
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Jul 2014
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I02-Macromolecular Crystallography
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Open Access
Abstract: The R2TP cochaperone complex plays a critical role in the assembly of multisubunit machines, including small nucleolar ribonucleoproteins (snoRNPs), RNA polymerase II, and the mTORC1 and SMG1 kinase complexes, but the molecular basis of substrate recognition remains unclear. Here, we describe a phosphopeptide binding domain (PIH-N) in the PIH1D1 subunit of the R2TP complex that preferentially binds to highly acidic phosphorylated proteins. A cocrystal structure of a PIH-N domain/TEL2 phosphopeptide complex reveals a highly specific phosphopeptide recognition mechanism in which Lys57 and 64 in PIH1D1, along with a conserved DpSDD phosphopeptide motif within TEL2, are essential and sufficient for binding. Proteomic analysis of PIH1D1 interactors identified R2TP complex substrates that are recruited by the PIH-N domain in a sequence-specific and phosphorylation-dependent manner suggestive of a common mechanism of substrate recognition. We propose that protein complexes assembled by the R2TP complex are defined by phosphorylation of a specific motif and recognition by the PIH1D1 subunit.
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Apr 2014
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I03-Macromolecular Crystallography
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David
Goldstone
,
Thomas G.
Flower
,
Neil J.
Ball
,
Marta
Sanz-Ramos
,
Melvyn
Yap
,
Roksana
Ogrodowicz
,
Nicole
Stanke
,
Juliane
Reh
,
Dirk
Lindemann
,
Jonathan P.
Stoye
,
Ian A.
Taylor
,
Christopher
Aiken
Open Access
Abstract: The Spumaretrovirinae, or foamyviruses (FVs) are complex retroviruses that infect many species of monkey and ape. Although FV infection is apparently benign, trans-species zoonosis is commonplace and has resulted in the isolation of the Prototypic Foamy Virus (PFV) from human sources and the potential for germ-line transmission.
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May 2013
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I04-Macromolecular Crystallography
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J. M.
Rock
,
D.
Lim
,
L.
Stach
,
R.
Ogrodowicz
,
J. M.
Keck
,
M. H.
Jones
,
C. C. L.
Wong
,
J. R.
Yates
,
M.
Winey
,
Stephen
Smerdon
,
M. B.
Yaffe
,
A.
Amon
Diamond Proposal Number(s):
[7707]
Abstract: Scaffold-assisted signaling cascades guide cellular decision-making. In budding yeast, one such signal transduction pathway called the mitotic exit network (MEN) governs the transition from mitosis to the G1 phase of the cell cycle. The MEN is conserved and in metazoans is known as the Hippo tumor-suppressor pathway. We found that signaling through the MEN kinase cascade was mediated by an unusual two-step process. The MEN kinase Cdc15 first phosphorylated the scaffold Nud1. This created a phospho-docking site on Nud1, to which the effector kinase complex Dbf2-Mob1 bound through a phosphoserine-threonine binding domain, in order to be activated by Cdc15. This mechanism of pathway activation has implications for signal transmission through other kinase cascades and might represent a general principle in scaffold-assisted signaling.
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Apr 2013
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