I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: The natural production of the β-lactam antibiotic carbapenem in bacteria involves a group of enzymes that form a synthetic pathway as well as proteins that protect the cell from self-intoxification by the products. Here, the crystal structure of CarF, one of the two proteins that confer resistance to synthesis of the antibiotic in the host organism, is reported. The CarF fold places it within a widely occurring structural family, indicating an ancient structural origin from which the resistance function has been derived.
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Jul 2017
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Hannes
Falke
,
Apirat
Chaikuad
,
Anja
Becker
,
Nadã¨ge
Loaec
,
Olivier
Lozach
,
Samira
Abu Jhaisha
,
Walter
Becker
,
Peter G.
Jones
,
Lutz
Preu
,
Knut
Baumann
,
Stefan
Knapp
,
Laurent
Meijer
,
Conrad
Kunick
Diamond Proposal Number(s):
[8421, 10619]
Open Access
Abstract: The protein kinase DYRK1A has been suggested to act as one of the intracellular regulators contributing to neurological alterations found in individuals with Down syndrome. For an assessment of the role of DYRK1A, selective synthetic inhibitors are valuable pharmacological tools. However, the DYRK1A inhibitors described in the literature so far either are not sufficiently selective or have not been tested against closely related kinases from the DYRK and the CLK protein kinase families. The aim of this study was the identification of DYRK1A inhibitors exhibiting selectivity versus the structurally and functionally closely related DYRK and CLK isoforms. Structure modification of the screening hit 11H-indolo[3,2-c]quinoline-6-carboxylic acid revealed structure–activity relationships for kinase inhibition and enabled the design of 10-iodo-substituted derivatives as very potent DYRK1A inhibitors with considerable selectivity against CLKs. X-ray structure determination of three 11H-indolo[3,2-c]quinoline-6-carboxylic acids cocrystallized with DYRK1A confirmed the predicted binding mode within the ATP binding site.
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Mar 2015
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I13-2-Diamond Manchester Imaging
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C. G.
Fenton
,
C. L.
Doig
,
S.
Fareed
,
A.
Naylor
,
A. P.
Morrell
,
O.
Addison
,
C.
Wehmeyer
,
C. D.
Buckley
,
M. S.
Cooper
,
G. G.
Lavery
,
K.
Raza
,
R. S.
Hardy
Diamond Proposal Number(s):
[16654]
Open Access
Abstract: Background: Despite their efficacy in the treatment of chronic inflammation, the prolonged application of therapeutic glucocorticoids (GCs) is limited by significant systemic side effects including glucocorticoid-induced osteoporosis (GIOP). 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a bi-directional enzyme that primarily activates GCs in vivo, regulating tissue-specific exposure to active GC. We aimed to determine the contribution of 11β-HSD1 to GIOP. Methods: Wild type (WT) and 11β-HSD1 knockout (KO) mice were treated with corticosterone (100 μg/ml, 0.66% ethanol) or vehicle (0.66% ethanol) in drinking water over 4 weeks (six animals per group). Bone parameters were assessed by micro-CT, sub-micron absorption tomography and serum markers of bone metabolism. Osteoblast and osteoclast gene expression was assessed by quantitative RT-PCR. Results: Wild type mice receiving corticosterone developed marked trabecular bone loss with reduced bone volume to tissue volume (BV/TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N). Histomorphometric analysis revealed a dramatic reduction in osteoblast numbers. This was matched by a significant reduction in the serum marker of osteoblast bone formation P1NP and gene expression of the osteoblast markers Alp and Bglap. In contrast, 11β-HSD1 KO mice receiving corticosterone demonstrated almost complete protection from trabecular bone loss, with partial protection from the decrease in osteoblast numbers and markers of bone formation relative to WT counterparts receiving corticosterone. Conclusions: This study demonstrates that 11β-HSD1 plays a critical role in GIOP, mediating GC suppression of anabolic bone formation and reduced bone volume secondary to a decrease in osteoblast numbers. This raises the intriguing possibility that therapeutic inhibitors of 11β-HSD1 may be effective in preventing GIOP in patients receiving therapeutic steroids.
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Aug 2019
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I02-Macromolecular Crystallography
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Open Access
Abstract: In tailed bacteriophages and several animal viruses, the portal protein forms the gateway through which viral DNA is translocated into the head structure during viral particle assembly. In the mature virion the portal protein exists as a dodecamer, while recombinant portal proteins from several phages, including SPP1 and CNPH82, have been shown to form 13-subunit assemblies. A putative portal protein from the thermostable bacteriophage G20C has been cloned, overexpressed and purified. Crystals of the protein diffracted to 2.1 Å resolution and belonged to space group P4212, with unit-cell parameters a = b = 155.3, c = 115.4 Å. The unit-cell content and self-rotation function calculations indicate that the protein forms a circular 12-subunit assembly.
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Nov 2013
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I04-Macromolecular Crystallography
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Christopher T.
Lohans
,
David Y.
Wang
,
Christian
Jorgensen
,
Samuel T.
Cahill
,
Ian J.
Clifton
,
Michael A.
Mcdonough
,
Henry P.
Oswin
,
James
Spencer
,
Carmen
Domene
,
Timothy D. W.
Claridge
,
Jurgen
Brem
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[12346]
Open Access
Abstract: The class D (OXA) serine β-lactamases are a major cause of resistance to β-lactam antibiotics. The class D enzymes are unique amongst β-lactamases because they have a carbamylated lysine that acts as a general acid/base in catalysis. Previous crystallographic studies led to the proposal that β-lactamase inhibitor avibactam targets OXA enzymes in part by promoting decarbamylation. Similarly, halide ions are proposed to inhibit OXA enzymes via decarbamylation. NMR analyses, in which the carbamylated lysines of OXA-10, -23 and -48 were 13C-labelled, indicate that reaction with avibactam does not ablate lysine carbamylation in solution. While halide ions did not decarbamylate the 13C-labelled OXA enzymes in the absence of substrate or inhibitor, avibactam-treated OXA enzymes were susceptible to decarbamylation mediated by halide ions, suggesting halide ions may inhibit OXA enzymes by promoting decarbamylation of acyl-enzyme complex. Crystal structures of the OXA-10 avibactam complex were obtained with bromide, iodide, and sodium ions bound between Trp-154 and Lys-70. Structures were also obtained wherein bromide and iodide ions occupy the position expected for the ‘hydrolytic water’ molecule. In contrast with some solution studies, Lys-70 was decarbamylated in these structures. These results reveal clear differences between crystallographic and solution studies on the interaction of class D β-lactamases with avibactam and halides, and demonstrate the utility of 13C-NMR for studying lysine carbamylation in solution.
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Jun 2017
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I07-Surface & interface diffraction
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Baocai
Du
,
Renyong
Geng
,
Wei
Li
,
Donghui
Li
,
Yuchao
Mao
,
Mengxue
Chen
,
Xue
Zhang
,
Joel A.
Smith
,
Rachel C.
Kilbride
,
Mary E.
O'Kane
,
Dan
Liu
,
David G.
Lidzey
,
Weihua
Tang
,
Tao
Wang
Diamond Proposal Number(s):
[22651]
Abstract: The insufficient phase separation between polymer donors and non-fullerene acceptors (NFAs) featuring with low-structural orders disrupts efficient charge transport and increases charge recombination, consequently limits the maximum achievable power conversion efficiency (PCE) of organic solar cells (OSCs). Herein, an NFA IT-M has been added as the third component into the PBDB-T:m-INPOIC OSCs, and is shown to effectively tune the phase separation between donor and acceptor molecules, although all components in the ternary system exhibit low degrees of structural orders. The incorporation of 10 wt% IT-M into a PBDB-T:m-INPOIC binary host blend appreciably increases the length scale of phase separation, creating continuous pathways which increase and balance charge transport. This leads to an enhanced photovoltaic performance from 12.8% in the binary cell to 13.9% for the ternary cell with simultaneously improved open-circuit voltage, short-circuit current and fill factor. This work highlights the beneficial role of ternary components in controlling the morphology of the active layer for high performance OSCs.
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Sep 2019
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I03-Macromolecular Crystallography
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Tobias
Karlberg
,
Peter
Hornyak
,
Ana Filipa
Pinto
,
Stefina
Milanova
,
Mahsa
Ebrahimi
,
Mikael
Lindberg
,
Nikolai
Püllen
,
Axel
Nordström
,
Elinor
Löverli
,
Rémi
Caraballo
,
Emily V.
Wong
,
Katja
Näreoja
,
Ann-Gerd
Thorsell
,
Mikael
Elofsson
,
Enrique M.
De La Cruz
,
Camilla
Björkegren
,
Herwig
Schüler
Diamond Proposal Number(s):
[11265, 15806]
Open Access
Abstract: Pseudomonas are a common cause of hospital-acquired infections that may be lethal. ADP-ribosyltransferase activities of Pseudomonas exotoxin-S and -T depend on 14-3-3 proteins inside the host cell. By binding in the 14-3-3 phosphopeptide binding groove, an amphipathic C-terminal helix of ExoS and ExoT has been thought to be crucial for their activation. However, crystal structures of the 14-3-3β:ExoS and -ExoT complexes presented here reveal an extensive hydrophobic interface that is sufficient for complex formation and toxin activation. We show that C-terminally truncated ExoS ADP-ribosyltransferase domain lacking the amphipathic binding motif is active when co-expressed with 14-3-3. Moreover, swapping the amphipathic C-terminus with a fragment from Vibrio Vis toxin creates a 14-3-3 independent toxin that ADP-ribosylates known ExoS targets. Finally, we show that 14-3-3 stabilizes ExoS against thermal aggregation. Together, this indicates that 14-3-3 proteins activate exotoxin ADP-ribosyltransferase domains by chaperoning their hydrophobic surfaces independently of the amphipathic C-terminal segment.
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Sep 2018
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I24-Microfocus Macromolecular Crystallography
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Philippe
Riou
,
Svend
Kjær
,
Ritu
Garg
,
Andy
Purkiss
,
Roger
George
,
Robert J.
Cain
,
Ganka
Bineva
,
Nicolas
Reymond
,
Brad
Mccoll
,
Andrew
Thompson
,
Nicola
O'Reilly
,
Neil
Mcdonald
,
Peter J.
Parker
,
Anne J.
Ridley
Diamond Proposal Number(s):
[8015]
Open Access
Abstract: Signaling through G proteins normally involves conformational switching between GTP- and GDP-bound states. Several Rho GTPases are also regulated by RhoGDI binding and sequestering in the cytosol. Rnd proteins are atypical constitutively GTP-bound Rho proteins, whose regulation remains elusive. Here, we report a high-affinity 14-3-3-binding site at the C terminus of Rnd3 consisting of both the Cys241-farnesyl moiety and a Rho-associated coiled coil containing protein kinase (ROCK)-dependent Ser240 phosphorylation site. 14-3-3 binding to Rnd3 also involves phosphorylation of Ser218 by ROCK and/or Ser210 by protein kinase C (PKC). The crystal structure of a phosphorylated, farnesylated Rnd3 peptide with 14-3-3 reveals a hydrophobic groove in 14-3-3 proteins accommodating the farnesyl moiety. Functionally, 14-3-3 inhibits Rnd3-induced cell rounding by translocating it from the plasma membrane to the cytosol. Rnd1, Rnd2, and geranylgeranylated Rap1A interact similarly with 14-3-3. In contrast to the canonical GTP/GDP switch that regulates most Ras superfamily members, our results reveal an unprecedented mechanism for G protein inhibition by 14-3-3 proteins.
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Apr 2013
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Giancarlo
Abis
,
Rebecca L.
Charles
,
Jolanta
Kopec
,
Wyatt W.
Yue
,
R. Andrew
Atkinson
,
Tam T. T.
Bui
,
Steven
Lynham
,
Simona
Popova
,
Yin-Biao
Sun
,
Franca
Fraternali
,
Philip
Eaton
,
Maria R.
Conte
Diamond Proposal Number(s):
[13597, 10619]
Open Access
Abstract: Human soluble epoxide hydrolase (hsEH) is an enzyme responsible for the inactivation of bioactive epoxy fatty acids, and its inhibition is emerging as a promising therapeutical strategy to target hypertension, cardiovascular disease, pain and insulin sensitivity. Here, we uncover the molecular bases of hsEH inhibition mediated by the endogenous 15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2). Our data reveal a dual inhibitory mechanism, whereby hsEH can be inhibited by reversible docking of 15d-PGJ2 in the catalytic pocket, as well as by covalent locking of the same compound onto cysteine residues C423 and C522, remote to the active site. Biophysical characterisations allied with in silico investigations indicate that the covalent modification of the reactive cysteines may be part of a hitherto undiscovered allosteric regulatory mechanism of the enzyme. This study provides insights into the molecular modes of inhibition of hsEH epoxy-hydrolytic activity and paves the way for the development of new allosteric inhibitors.
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May 2019
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I11-High Resolution Powder Diffraction
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Diamond Proposal Number(s):
[10038]
Open Access
Abstract: Low-pH cements are candidate materials for use in the construction of geological disposal facilities for the long-term management of nuclear waste. Since these facilities will operate over long time scales, the changes in mineralogy and microstructure require evaluation as a function of time. As a first step towards this understanding, the hydration of a standardised low-pH cement paste, known as the Cebama reference cement, was investigated over an 18-month period. Characterisation was performed at 28 days of curing, at 20 °C and 40 °C, and novel synchrotron radiation X-ray diffraction experiments were performed, in-situ, from 90 min to 18 months of curing. Concurrent solid state 29Si and 27Al MAS NMR data were acquired for parallel samples to quantify the extent of cement hydration and the composition and mean chain length of the predominant calcium aluminosilicate hydrate (C-(A)-S-H) reaction product. After 18 months, cement clinker phases were still present, highlighting the slow hydration kinetics of this low-pH cement. The data presented provide a benchmark for ongoing and future studies of low-pH cements in geological disposal environments, over extended time scales.
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Mar 2020
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