I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: Fragment based methods are now widely used to identify starting points in drug discovery and generation of tools for chemical biology. A significant challenge is optimization of these weak binding fragments to hit and lead compounds. We have developed an approach where individual reaction mixtures of analogues of hits can be evaluated without purification of the product. Here, we describe experiments to optimise the processes and then assess such mixtures in the high throughput crystal structure determination facility, XChem. Diffraction data for crystals of the proteins Hsp90 and PDHK2 soaked individually with 83 crude reaction mixtures are analysed manually or with the automated XChem procedures. The results of structural analysis are compared with binding measurements from other biophysical techniques. This approach can transform early hit to lead optimisation and the lessons learnt from this study provide a protocol that can be used by the community.
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Sep 2020
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B21-High Throughput SAXS
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15868, 22906]
Abstract: In tryptophan biosynthesis, the reaction catalyzed by the enzyme indole-3-glycerol phosphate synthase (IGP synthase, IGPS) starts with a condensation step where the substrate’s carboxylated phenyl group makes a nucleophilic attack to form the pyrrole ring of the indole, followed by a decarboxylation which restores the aromaticity of the phenyl. IGPS from Pseudomonas aeruginosa has the highest turnover number of all characterized IGPS enzymes, providing an excellent model system to test the necessity of the decarboxylation step. This step was since the 1960s considered to be mechanistically essential based on studies of the IGPS:PRAI fusion protein from Escherichia coli. Here, we present the crystal structure of P. aeruginosa IGPS in complex with reduced CdRP, a non-reactive substrate analogue, and using a sensitive discontinuous assay, we demonstrate weak promiscuous activity on the decarboxylated substrate 1-(phenylamino)-1-deoxyribulose-5-phosphate (PAdRP) – with approximately 1000' lower rate of IGP formation than from the native substrate. We also show that E. coli IGPS, at even lower rate, can produce IGP from decarboxylated substrate. Our structure of P. aeruginosa IGPS has eight molecules in the asymmetric unit, out of which seven contain ligand, and one displays a previously unobserved conformation closer to the reactive state. One of the few non-conserved active-site residues, Phe201 in P. aeruginosa IGPS, is by mutagenesis demonstrated to be important for the higher turnover of this enzyme on both substrates. Our results demonstrate that, despite IGPS’s classification as a carboxylyase (i.e. decarboxylase), decarboxylation not a completely essential step in its catalysis.
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Sep 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: Ribonucleotide reductase (RNR) is a central enzyme for DNA building block synthesis. Most aerobic organisms, including nearly all eukaryotes, have class I RNRs consisting of R1 and R2 subunits. The catalytic R1 subunit contains an overall activity site that can allosterically turn the enzyme on or off by the binding of ATP or dATP, respectively. The mechanism behind the ability to turn the enzyme off via the R1 subunit involves the formation of different types of R1 oligomers in most studied species and R1-R2 octamers in Escherichia coli. To better understand the distribution of different oligomerization mechanisms, we characterized the enzyme from Clostridium botulinum, which belongs to a subclass of class I RNRs not studied before. The recombinantly expressed enzyme was analyzed by size exclusion chromatography, gas-phase electrophoretic mobility macromolecular analysis, electron microscopy, x-ray crystallography, and enzyme assays. Interestingly, it shares the ability of the E. coli RNR to form inhibited R1-R2 octamers in the presence of dATP but, unlike the E. coli enzyme, cannot be turned off by combinations of ATP and dGTP/dTTP. A phylogenetic analysis of class I RNRs suggests that activity regulation is not ancestral, but was gained after the first subclasses diverged and that RNR subclasses with inhibition mechanisms involving R1 oligomerization belong to a clade separated from the two subclasses forming R1-R2 octamers. These results give further insight into activity regulation in class I RNRs as an evolutionarily dynamic process.
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Sep 2020
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Open Access
Abstract: Autophagy is a common name for a number of catabolic processes, which keep the cellular homeostasis by removing damaged and dysfunctional intracellular components. Impairment or misbalance of autophagy can lead to various diseases, such as neurodegeneration, infection diseases, and cancer. A central axis of autophagy is formed along the interactions of autophagy modifiers (Atg8-family proteins) with a variety of their cellular counter partners. Besides autophagy, Atg8-proteins participate in many other pathways, among which membrane trafficking and neuronal signaling are the most known. Despite the fact that autophagy modifiers are well-studied, as the small globular proteins show similarity to ubiquitin on a structural level, the mechanism of their interactions are still not completely understood. A thorough analysis and classification of all known mechanisms of Atg8-protein interactions could shed light on their functioning and connect the pathways involving Atg8-proteins. In this review, we present our views of the key features of the Atg8-proteins and describe the basic principles of their recognition and binding by interaction partners. We discuss affinity and selectivity of their interactions as well as provide perspectives for discovery of new Atg8-interacting proteins and therapeutic approaches to tackle major human diseases.
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Sep 2020
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Yanchun
Peng
,
Alexander J.
Mentzer
,
Guihai
Liu
,
Xuan
Yao
,
Zixi
Yin
,
Danning
Dong
,
Wanwisa
Dejnirattisai
,
Timothy
Rostron
,
Piyada
Supasa
,
Chang
Liu
,
César
López-camacho
,
Jose
Slon-campos
,
Yuguang
Zhao
,
David I.
Stuart
,
Guido C.
Paesen
,
Jonathan M.
Grimes
,
Alfred A.
Antson
,
Oliver W.
Bayfield
,
Dorothy E. D. P.
Hawkins
,
De-sheng
Ker
,
Beibei
Wang
,
Lance
Turtle
,
Krishanthi
Subramaniam
,
Paul
Thomson
,
Ping
Zhang
,
Christina
Dold
,
Jeremy
Ratcliff
,
Peter
Simmonds
,
Thushan
De Silva
,
Paul
Sopp
,
Dannielle
Wellington
,
Ushani
Rajapaksa
,
Yi-ling
Chen
,
Mariolina
Salio
,
Giorgio
Napolitani
,
Wayne
Paes
,
Persephone
Borrow
,
Benedikt M.
Kessler
,
Jeremy W.
Fry
,
Nikolai F.
Schwabe
,
Malcolm G.
Semple
,
J. Kenneth
Baillie
,
Shona C.
Moore
,
Peter J. M.
Openshaw
,
M. Azim
Ansari
,
Susanna
Dunachie
,
Eleanor
Barnes
,
John
Frater
,
Georgina
Kerr
,
Oliver
Gould
,
Teresa
Lockett
,
Robert
Levin
,
Yonghong
Zhang
,
Ronghua
Jing
,
Ling-pei
Ho
,
Richard J.
Cornall
,
Christopher P.
Conlon
,
Paul
Klenerman
,
Gavin R.
Screaton
,
Juthathip
Mongkolsapaya
,
Andrew
Mcmichael
,
Julian C.
Knight
,
Graham
Ogg
,
Tao
Dong
Open Access
Abstract: The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases. Total and spike-specific T cell responses correlated with spike-specific antibody responses. We identified 41 peptides containing CD4+ and/or CD8+ epitopes, including six immunodominant regions. Six optimized CD8+ epitopes were defined, with peptide–MHC pentamer-positive cells displaying the central and effector memory phenotype. In mild cases, higher proportions of SARS-CoV-2-specific CD8+ T cells were observed. The identification of T cell responses associated with milder disease will support an understanding of protective immunity and highlights the potential of including non-spike proteins within future COVID-19 vaccine design.
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Sep 2020
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I13-2-Diamond Manchester Imaging
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Open Access
Abstract: Hypothesis: We define contact angles, h, during displacement of three fluid phases in a porous medium using energy balance, extending previous work on two-phase flow. We test if this theory can be applied to quantify the three contact angles and wettability order in pore-scale images of three-phase displacement. Theory: For three phases labelled 1, 2 and 3, and solid, s, using conservation of energy ignoring viscous dissipation ðDa1s cos h12 Da12 /j12 DS1 Þr12 1⁄4 ðDa3s cos h23 þ Da23 /j23 DS3 Þr23 þ Da13 r13 , where / is the porosity, r is the interfacial tension, a is the specific interfacial area, S is the saturation, and j is the fluid–fluid interfacial curvature. D represents the change during a displacement. The third contact angle, h13 can be found using the Bartell-Osterhof relationship. The energy balance is also extended to an arbitrary number of phases.
Findings: X-ray imaging of porous media and the fluids within them, at pore-scale resolution, allows the difference terms in the energy balance equation to be measured. This enables wettability, the contact angles, to be determined for complex displacements, to characterize the behaviour, and for input into pore-scale models. Two synchrotron imaging datasets are used to illustrate the approach, comparing the flow of oil, water and gas in a water-wet and an altered-wettability limestone rock sample. We show that in the water-wet case, as expected, water (phase 1) is the most wetting phase, oil (phase 2) is inter- mediate wet, while gas (phase 3) is most non-wetting with effective contact angles of h12 % 48 and h13 % 44, while h23 1⁄4 0 since oil is always present in spreading layers. In contrast, for the altered-wettability case, oil is most wetting, gas is intermediate-wet, while water is most non-wetting with con- tact angles of h12 1⁄4 134 $ 10;h13 1⁄4 119 $ 10, and h23 1⁄4 66 $ 10.
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Sep 2020
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Open Access
Abstract: Hard X-ray nanotomography enables 3D investigations of a wide range of samples with high resolution (<100 nm) with both synchrotron-based and laboratory-based setups. However, the advantage of synchrotron-based setups is the high flux, enabling time resolution, which cannot be achieved at laboratory sources. Here, the nanotomography setup at the imaging beamline P05 at PETRA III is presented, which offers high time resolution not only in absorption but for the first time also in Zernike phase contrast. Two test samples are used to evaluate the image quality in both contrast modalities based on the quantitative analysis of contrast-to-noise ratio (CNR) and spatial resolution. High-quality scans can be recorded in 15 min and fast scans down to 3 min are also possible without significant loss of image quality. At scan times well below 3 min, the CNR values decrease significantly and classical image-filtering techniques reach their limitation. A machine-learning approach shows promising results, enabling acquisition of a full tomography in only 6 s. Overall, the transmission X-ray microscopy instrument offers high temporal resolution in absorption and Zernike phase contrast, enabling in situ experiments at the beamline.
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Sep 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[12788, 17773]
Open Access
Abstract: Reductive dehalogenases are responsible for the reductive cleavage of carbon-halogen bonds during organohalide respiration. A variety of mechanisms have been proposed for these cobalamin and [4Fe-4S] containing enzymes, including organocobalt, radical, or cobalt-halide adduct based catalysis. The latter was proposed for the oxygen-tolerant Nitratireductor pacificus pht-3B catabolic reductive dehalogenase (NpRdhA). Here, we present the first substrate bound NpRdhA crystal structures, confirming a direct cobalt–halogen interaction is established and providing a rationale for substrate preference. Product formation is observed in crystallo due to X-ray photoreduction. Protein engineering enables rational alteration of substrate preference, providing a future blue print for the application of this and related enzymes in bioremediation.
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Sep 2020
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I20-Scanning-X-ray spectroscopy (XAS/XES)
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Diamond Proposal Number(s):
[19013]
Open Access
Abstract: Surfactant-mediated chemical routes allow one to synthesize highly engineered shape- and size-controlled nanocrystals. However, the occurrence of capping agents on the surface of the nanocrystals is undesirable for selected applications. Here, a novel approach to the production of shape-controlled nanocrystals which exhibit high thermal stability is demonstrated. Ceria nanocubes obtained by surfactant-mediated synthesis are embedded inside a highly porous silica aerogel and thermally treated to remove the capping agent. Powder X-ray Diffraction and Scanning Transmission Electron Microscopy show the homogeneous dispersion of the nanocubes within the aerogel matrix. Remarkably, both the size and the shape of the ceria nanocubes are retained not only throughout the aerogel syntheses but also upon thermal treatments up to 900 °C, while avoiding their agglomeration. The reactivity of ceria is measured by in situ High-Energy Resolution Fluorescence Detected - X-ray Absorption Near Edge Spectroscopy at the Ce L3 edge, and shows the reversibility of redox cycles of ceria nanocubes when they are embedded in the aerogel. This demonstrates that the enhanced reactivity due to their prominent {100} crystal facets is preserved. In contrast, unsupported ceria nanocubes begin to agglomerate as soon as the capping agent decomposes, leading to a degradation of their reactivity already at 275 °C.
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Sep 2020
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B18-Core EXAFS
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Diamond Proposal Number(s):
[15151]
Open Access
Abstract: The activation of C–H bonds for carbon–carbon coupling reactions remains a challenge in organic synthesis. Visible light photocatalysis offers a unique opportunity to sustainably perform these reactions in a single-step, without the need for caustic reagents, and under ambient operating conditions. We utilize non-noble metals in the form of hybrid cobalt-based zeolitic imidazole frameworks, for the first time, to explore the structure–property correlations leading to the photocatalytic formation of C–C bonds. Combining in situ spectroscopy and theoretical simulations we can rationalize the photocatalytic efficacy of different frameworks. This led to an improved understanding of the nature of the photocatalytic active sites and associated reaction pathway.
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Sep 2020
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