I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Larissa
Romanello
,
Ana Eliza
Zeraik
,
Adriano
De Freitas Fernandes
,
Juliana Roberta
Torini
,
Louise E.
Bird
,
Joanne E.
Nettleship
,
Heather
Rada
,
Yamini
Reddivari
,
Ray J.
Owens
,
Vitor Hugo Balasco
Serrão
,
Ricardo
Demarco
,
Jose
Brandao-neto
,
Humberto
D'muniz Pereira
Diamond Proposal Number(s):
[5073]
Abstract: Schistosoma mansoni, the parasite responsible for schistosomiasis, lacks the “de novo” purine biosynthetic pathway and depends entirely on the purine salvage pathway for the supply of purines. Numerous reports of praziquantel resistance have been described, as well as stimulated efforts to develop new drugs against schistosomiasis. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme of the purine salvage pathway. Here, we describe a crystallographic structure of the S. mansoni HPGRT-1 (SmHGPRT), complexed with IMP at a resolution of 2.8Ǻ. Four substitutions were identified in the region of the active site between SmHGPRT-1 and human HGPRT. We also present data from RNA-Seq and WISH, suggesting that some isoforms of HGPRT might be involved in the process related to sexual maturation and reproduction in worms; furthermore, its enzymatic assays show that the isoform SmHGPRT-3 does not present the same catalytic efficiency as other isoforms. Finally, although other studies have previously suggested this enzyme as a potential antischistosomal chemotherapy target, the kinetics parameters reveal the impossibility to use SmHGPRT as an efficient chemotherapeutic target.
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Feb 2019
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14744]
Abstract: cAMP response element binding protein 3 (CREB3) is an endoplasmic reticulum (ER) membrane‐bound transcription factor, which belongs to the basic leucine zipper (bZIP) superfamily of eukaryotic transcription factors. CREB3 plays a role in the ER‐stress induced unfolded protein response (UPR) and is a multifunctional cellular factor implicated in a number of biological processes including cell proliferation and migration, tumour suppression, and immune‐related gene expression. To gain structural insights into the transcription factor, we determined the crystal structure of the conserved bZIP domain of chicken CREB3 (chCREB3) to a resolution of 3.95Å. The X‐ray structure provides evidence that chCREB3 can form a stable homodimer. The chCREB3 bZIP has a structured, pre‐formed DNA binding region, even in the absence of DNA, a feature that could potentially enhance both the DNA binding specificity and affinity of chCREB3. Significantly, the homodimeric bZIP possesses an intermolecular disulphide bond that connects equivalent cysteine residues of the parallel helices in the leucine zipper region. This disulphide bond in the hydrophobic core of the bZIP may increase the stability of the homodimer under oxidising conditions. Moreover, sequence alignment of bZIP sequences from chicken, human and mouse reveals only members of the CREB3 subfamily contain this cysteine residue, indicating it could act as a redox‐sensor. Taken together, these results suggest activity of these transcription factors may be redox‐regulated and they may be activated in response to oxidative stress.
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Jan 2019
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Ramya
Salimraj
,
Philip
Hinchliffe
,
Magda
Kosmopoulou
,
Jonathan M.
Tyrrell
,
Jurgen
Brem
,
Sander S.
Van Berkel
,
Anil
Verma
,
Raymond J.
Owens
,
Michael A.
Mcdonough
,
Timothy R.
Walsh
,
Christopher J.
Schofield
,
James
Spencer
Diamond Proposal Number(s):
[313]
Abstract: Metallo‐β‐Lactamases (MBLs) protect bacteria from almost all β‐lactam antibiotics. VIM enzymes are among the most clinically important MBLs, with VIM‐1 increasing in carbapenem‐resistant Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae) that are amongst the hardest bacterial pathogens to treat. VIM enzymes display sequence variation at residues (224 and 228) that in related MBLs are conserved and participate in substrate binding. How they accommodate this variability, while retaining catalytic efficiency against a broad substrate range, has remained unclear. Here we present crystal structures of VIM‐1 and its complexes with a substrate‐mimicking thioenolate inhibitor, ML302F, that restores meropenem activity against a range of VIM‐1 producing clinical strains, and the hydrolysed product of the carbapenem meropenem. Comparison of these two structures identifies a water‐mediated hydrogen bond, between the carboxylate group of substrate/inhibitor and the backbone carbonyl of the active site zinc ligand Cys221, that is common to both complexes. Structural comparisons show that the responsible Cys221‐bound water is observed in all known VIM structures, participates in carboxylate binding with other inhibitor classes, and thus effectively replicates the role of the conserved Lys224 in analogous complexes with other MBLs. These results provide a mechanism for substrate binding that permits the variation at positions 224 and 228 that is a hallmark of VIM MBLs.
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Nov 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Juliana Roberta
Torini
,
Larissa
Romanello
,
Fernanda Aparecida Heleno
Batista
,
Vitor Hugo Balasco
Serrao
,
Muhamamd
Faheem
,
Ana Eliza
Zeraik
,
Louise
Bird
,
Joanne E.
Nettleship
,
Yamini
Reddivari
,
Ray
Owens
,
Ricardo
Demarco
,
Júlio César
Borges
,
Jose
Brandao-neto
,
Humberto
D'muniz Pereira
Open Access
Abstract: Purine nucleoside phosphorylases (PNPs) play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway. Here we present the structural and kinetic characterization of a new PNP isoform from S. mansoni, SmPNP2. Thermofluorescence screening of different ligands suggested cytidine and cytosine are potential ligands. The binding of cytosine and cytidine were confirmed by isothermal titration calorimetry, with a KD of 27 μM for cytosine, and a KM of 76.3 μM for cytidine. SmPNP2 also displays catalytic activity against inosine and adenosine, making it the first described PNP with robust catalytic activity towards both pyrimidines and purines. Crystal structures of SmPNP2 with different ligands were obtained and comparison of these structures with the previously described S. mansoni PNP (SmPNP1) provided clues for the unique capacity of SmPNP2 to bind pyrimidines. When compared with the structure of SmPNP1, substitutions in the vicinity of SmPNP2 active site alter the architecture of the nucleoside base binding site thus permitting an alternative binding mode for nucleosides, with a 180° rotation from the canonical binding mode. The remarkable plasticity of this binding site enhances our understanding of the correlation between structure and nucleotide selectivity, thus suggesting new ways to analyse PNP activity.
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Sep 2018
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Gareth
Shimmon
,
Abhay
Kotecha
,
Jingshan
Ren
,
Amin S.
Asfor
,
Joseph
Newman
,
Stephen
Berryman
,
Eleanor M.
Cottam
,
Sarah
Gold
,
Toby J.
Tuthill
,
Donald P.
King
,
Emiliana
Brocchi
,
Andrew M. Q.
King
,
Ray
Owens
,
Elizabeth E.
Fry
,
David I.
Stuart
,
Alison
Burman
,
Terry
Jackson
Open Access
Abstract: Foot-and-mouth disease (FMD) affects economically important livestock and is one of the most contagious viral diseases. The most commonly used FMD diagnostic assay is a sandwich ELISA. However, the main disadvantage of this ELISA is that it requires anti-FMD virus (FMDV) serotype-specific antibodies raised in small animals. This problem can be, in part, overcome by using anti-FMDV monoclonal antibodies (MAbs) as detecting reagents. However, the long-term use of MAbs may be problematic and they may need to be replaced. Here we have constructed chimeric antibodies (mouse/rabbit D9) and Fabs (fragment antigen-binding) (mouse/cattle D9) using the Fv (fragment variable) regions of a mouse MAb, D9 (MAb D9), which recognises type O FMDV. The mouse/rabbit D9 chimeric antibody retained the FMDV serotype-specificity of MAb D9 and performed well in a FMDV detection ELISA as well as in routine laboratory assays. Cryo-electron microscopy analysis confirmed engagement with antigenic site 1 and peptide competition studies identified the aspartic acid at residue VP1 147 as a novel component of the D9 epitope. This chimeric expression approach is a simple but effective way to preserve valuable FMDV antibodies, and has the potential for unlimited generation of antibodies and antibody fragments in recombinant systems with the concomitant positive impacts on the 3Rs (Replacement, Reduction and Refinement) principles.
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Aug 2018
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I24-Microfocus Macromolecular Crystallography
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James I.
Robinson
,
Euan W.
Baxter
,
Robin L.
Owen
,
Maren
Thomsen
,
Darren C.
Tomlinson
,
Mark P.
Waterhouse
,
Stephanie J.
Win
,
Joanne E.
Nettleship
,
Christian
Tiede
,
Richard J.
Foster
,
Raymond J.
Owens
,
Colin W. G.
Fishwick
,
Sarah A.
Harris
,
Adrian
Goldman
,
Michael J.
Mcpherson
,
Ann W.
Morgan
Diamond Proposal Number(s):
[5969]
Abstract: Protein–protein interactions are essential for the control of cellular functions and are critical for regulation of the immune system. One example is the binding of Fc regions of IgG to the Fc gamma receptors (FcγRs). High sequence identity (98%) between the genes encoding FcγRIIIa (expressed on macrophages and natural killer cells) and FcγRIIIb (expressed on neutrophils) has prevented the development of monospecific agents against these therapeutic targets. We now report the identification of FcγRIIIa-specific artificial binding proteins called “Affimer” that block IgG binding and abrogate FcγRIIIa-mediated downstream effector functions in macrophages, namely TNF release and phagocytosis. Cocrystal structures and molecular dynamics simulations have revealed the structural basis of this specificity for two Affimer proteins: One binds directly to the Fc binding site, whereas the other acts allosterically.
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Jan 2018
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I02-Macromolecular Crystallography
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Larissa
Romanello
,
Vitor Hugo Balasco
Serrao
,
Juliana Roberta Torini
De Souza
,
Louise E.
Bird
,
Joanne E.
Nettleship
,
Heather
Rada
,
Yamini
Reddivari
,
Ray J.
Owens
,
Ricardo
De Marco
,
Jose
Brandao-neto
,
Humberto
D' Muniz Pereira
Diamond Proposal Number(s):
[5073, 5894]
Abstract: Schistosoma mansoni is the parasite responsible for schistosomiasis, a disease that affects about 218 million people worldwide. Currently, both direct treatment and disease control initiatives rely on chemotherapy using a single drug, praziquantel. Concerns over the possibility of resistance developing to praziquantel, have stimulated efforts to develop new drugs for the treatment of schistosomiasis. Schistosomes do not have the de novo purine biosynthetic pathway, and instead depend entirely on the purine salvage pathway to supply its need for purines. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. Adenylosuccinate lyase (SmADSL) is an enzyme in this pathway, which cleaves adenylosuccinate (ADS) into adenosine 5'-monophosphate (AMP) and fumarate. SmADSL kinetic characterization was performed by isothermal titration calorimetry (ITC) using both ADS and SAICAR as substrates. Structures of SmADSL in Apo form and in complex with AMP were elucidated by x-ray crystallography revealing a highly conserved tetrameric structure required for their function since the active sites is formed from residues of three different subunits. The active sites are also highly conserved between species and it is difficult to identify a potent species-specific inhibitor for the development of new therapeutic agents. In contrast, several mutagenesis studies have demonstrated the importance of dimeric interface residues in the stability of the quaternary structure of the enzyme. The lower conservation of these residues between SmADSL and human ADSL could be used to lead the development of anti-schistosomiasis drugs based on disruption of subunit interfaces. These structures and kinetics data add another layer of information to Schistosoma mansoni purine salvage pathway.
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Mar 2017
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I04-Macromolecular Crystallography
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Martin
Roatsch
,
Dina
Robaa
,
Martin
Pippel
,
Joanne E
Nettleship
,
Yamini
Reddivari
,
Louise
Bird
,
Inga
Hoffmann
,
Henriette
Franz
,
Ray
Owens
,
Roland
Schüle
,
Ralf
Flaig
,
Wolfgang
Sippl
,
Manfred
Jung
Open Access
Abstract: Aberrant expression of iron(II)- and 2-oxoglutarate-dependent JumonjiC histone demethylases has been linked to cancer. Potent demethylase inhibitors are drug candidates and biochemical tools to elucidate the functional impact of demethylase inhibition.
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Mar 2016
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E. W
Baxter
,
J. I
Robinson
,
D. C
Tomlinson
,
R. J
Foster
,
R. L
Owen
,
S. J
Win
,
J. E
Nettleship
,
C
Tiede
,
J
Kankanala
,
R. J
Owens
,
C. W. G
Fishwick
,
M. J
Mcpherson
,
A. W
Morgan
Abstract: Protein-protein interactions are essential for the control of cellular functions and critical for regulation of the immune system. One example is the binding of Fc regions of Immunoglobulin G to their receptors (Fcγ Receptors). High sequence identity (98%) between the genes encoding FcγRIIIa and FcγRIIIb has led to the lack of specific agents against this important therapeutic target. We aimed to develop a novel drug development pipeline using artificial binding proteins called Adhirons both for the identification of novel therapeutics and to guide drug discovery through the identification of novel hot spots/ druggable surfaces on the receptor.
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Feb 2016
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: Penicillin-binding protein 3 (PBP3) from Pseudomonas aeruginosa is themolecular target of b-lactam-based antibiotics. Structures of PBP3 in com-plexes with azlocillin and cefoperazone, which are in clinical use for the treat-ment of pseudomonad infections, have been determined to 2.0A resolution.Together with data from other complexes, these structures identify a commonset of residues involved in the binding of b-lactams to PBP3. Comparison ofwild-type and an active site mutant (S294A) showed that increased thermalstability of PBP3 following azlocillin binding was entirely due to covalentbinding to S294, whereas cefoperazone binding produces some increase in sta-bility without the covalent link. Consistent with this, a third crystal structurewas determined in which the hydrolysis product of cefoperazone was noncova-lently bound in the active site of PBP3. This is the first structure of a com-plex between a penicillin-binding protein and cephalosporic acid and may beimportant in the design of new noncovalent PBP3 inhibitors.
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Jan 2016
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