scholarly journals Fragment binding to the Nsp3 macrodomain of SARS-CoV-2 identified through crystallographic screening and computational docking

2021 ◽  
Vol 7 (16) ◽  
pp. eabf8711
Author(s):  
Marion Schuller ◽  
Galen J. Correy ◽  
Stefan Gahbauer ◽  
Daren Fearon ◽  
Taiasean Wu ◽  
...  
Keyword(s):  
Active Site ◽  
Nonstructural Protein ◽  
Adenosine Diphosphate ◽  
Titration Calorimetry ◽  
Conformational Heterogeneity ◽  
Time Resolved ◽  
Computational Docking ◽  
Wide Range ◽  
Differential Scanning Fluorimetry ◽  
Biophysical Techniques

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate–ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

scholarly journals Fragment Binding to the Nsp3 Macrodomain of SARS-CoV-2 Identified Through Crystallographic Screening and Computational Docking

2020 ◽  
Author(s):  
Marion Schuller ◽  
Galen J. Correy ◽  
Stefan Gahbauer ◽  
Daren Fearon ◽  
Taiasean Wu ◽  
...  
Keyword(s):  
High Resolution ◽  
Active Site ◽  
Conformational Heterogeneity ◽  
Structural Protein ◽  
Computational Docking ◽  
X Ray ◽  
Wide Range ◽  
Biophysical Techniques ◽  
Fragment Libraries ◽  
Chemical Matter

ABSTRACTThe SARS-CoV-2 macrodomain (Mac1) within the non-structural protein 3 (Nsp3) counteracts host-mediated antiviral ADP-ribosylation signalling. This enzyme is a promising antiviral target because catalytic mutations render viruses non-pathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of diverse fragment libraries resulted in 214 unique macrodomain-binding fragments, out of 2,683 screened. An additional 60 molecules were selected from docking over 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several crystallographic and docking fragment hits were validated for solution binding using three biophysical techniques (DSF, HTRF, ITC). Overall, the 234 fragment structures presented explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

scholarly journals Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Daniel A Keedy ◽  
Lillian R Kenner ◽  
Matthew Warkentin ◽  
Rahel A Woldeyes ◽  
Jesse B Hopkins ◽  
...  
Keyword(s):  
Active Site ◽  
Protein Function ◽  
Conformational Ensemble ◽  
Conformational Heterogeneity ◽  
Time Resolved ◽  
Protein Motions ◽  
Catalytic Function ◽  
Temperature Dependences ◽  
Site Network ◽  
Response To Temperature

Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180-–240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.

scholarly journals Mass spectrometry for fragment screening

2017 ◽  
Vol 61 (5) ◽  
pp. 465-473 ◽  
Author(s):  
Daniel Shiu-Hin Chan ◽  
Andrew J. Whitehouse ◽  
Anthony G. Coyne ◽  
Chris Abell
Keyword(s):  
Drug Discovery ◽  
High Sensitivity ◽  
Titration Calorimetry ◽  
Label Free ◽  
X Ray ◽  
X Ray Crystallography ◽  
Fragment Screening ◽  
Differential Scanning Fluorimetry ◽  
Biophysical Techniques

Fragment-based approaches in chemical biology and drug discovery have been widely adopted worldwide in both academia and industry. Fragment hits tend to interact weakly with their targets, necessitating the use of sensitive biophysical techniques to detect their binding. Common fragment screening techniques include differential scanning fluorimetry (DSF) and ligand-observed NMR. Validation and characterization of hits is usually performed using a combination of protein-observed NMR, isothermal titration calorimetry (ITC) and X-ray crystallography. In this context, MS is a relatively underutilized technique in fragment screening for drug discovery. MS-based techniques have the advantage of high sensitivity, low sample consumption and being label-free. This review highlights recent examples of the emerging use of MS-based techniques in fragment screening.

Characterization of P. aeruginosa Glucose 6- Phosphate Isomerase: A Functional Insight via In-Vitro Activity Study

2020 ◽  
Vol 20 (29) ◽  
pp. 2651-2661
Author(s):  
Deekshi Angira ◽  
Nalini Natarajan ◽  
Samir R. Dedania ◽  
Darshan H. Patel ◽  
Vijay Thiruvenkatam
Keyword(s):  
Active Site ◽  
Expression System ◽  
Homology Model ◽  
Competitive Inhibitor ◽  
Membered Ring ◽  
Titration Calorimetry ◽  
Motility Factor ◽  
Differential Scanning Fluorimetry ◽  
Ic50 Values

Background: Glucose-6-phosphate isomerase (G6PI) catalyses the second step in glycolysis in the reversible interconversion of an aldohexose glucose 6-phosphate, a six membered ring moiety to a ketohexose, fructose 6-phosphate five membered ring moiety. This enzyme is of utmost importance due to its multifunctional role like neuroleukin, autocrine motility factor, etc. in various species. G6PI from Pseudomonas aeruginosa is less explored for its moonlighting properties. These properties can be predicted by studying the active site conservation of residues and their interaction with the specific ligand. Methods: Here, we study the G6PI in a self-inducible construct in bacterial expression system with its purification using Ni-NTA chromatography. The secondary structure of pure G6PI is estimated using circular dichroism to further predict the proper folding form of the protein. The bioactivity of the purified enzyme is quantified using phosphoglucose isomerase colorimetric kit with a value of 12.5 mU/mL. Differential scanning fluorimetry and isothermal titration calorimetry were employed to monitor the interaction of G6PI with its competitive inhibitor, erythrose 4-phosphate and calculated the Tm, Kd and IC50 values. Further, the homology model for the protein was prepared to study the interaction with the erythrose 4-phosphate. MD simulation of the complex was performed at 100 ns to identify the binding interactions. Results: We identified hydrogen bonds and water bridges dominating the interactions in the active site holding the protein and ligand with strong affinity. Conclusion : G6PI was successfully crystallized and data has been collected at 6Å. We are focused on improving the crystal quality for obtaining higher resolution data.

Reduction of carbon impurities in aluminium nitride from time-resolved chemical vapour deposition using trimethylaluminum

2020 ◽  
Author(s):  
Polla Rouf ◽  
Pitsiri Sukkaew ◽  
Lars Ojamäe ◽  
Henrik Pedersen
Keyword(s):  
Chemical Vapour Deposition ◽  
Vapour Deposition ◽  
Chemical Vapour ◽  
Aluminium Nitride ◽  
Methyl Groups ◽  
Time Resolved ◽  
Thermally Induced ◽  
Light Emitting ◽  
Carbon Impurities ◽  
Wide Range

<p>Aluminium nitride (AlN) is a semiconductor with a wide range of applications from light emitting diodes to high frequency transistors. Electronic grade AlN is routinely deposited at 1000 °C by chemical vapour deposition (CVD) using trimethylaluminium (TMA) and NH<sub>3</sub> while low temperature CVD routes to high quality AlN are scarce and suffer from high levels of carbon impurities in the film. We report on an ALD-like CVD approach with time-resolved precursor supply where thermally induced desorption of methyl groups from the AlN surface is enhanced by the addition of an extra pulse, H<sub>2</sub>, N<sub>2</sub> or Ar between the TMA and NH<sub>3</sub> pulses. The enhanced desorption allowed deposition of AlN films with carbon content of 1 at. % at 480 °C. Kinetic- and quantum chemical modelling suggest that the extra pulse between TMA and NH<sub>3</sub> prevents re-adsorption of desorbing methyl groups terminating the AlN surface after the TMA pulse. </p>

Strongly Coupled Redox-Linked Conformational Switching at the Active Site of the Non-Heme Iron-Dependent Dioxygenase, TauD

2019 ◽  
Author(s):  
Christopher John ◽  
Greg M. Swain ◽  
Robert P. Hausinger ◽  
Denis A. Proshlyakov
Keyword(s):  
Active Site ◽  
Structural Model ◽  
Heme Iron ◽  
Chemical Transformations ◽  
Experimental Conditions ◽  
Strongly Coupled ◽  
Non Heme Iron ◽  
Reversible Redox ◽  
Wide Range ◽  
Complex Structural

2-Oxoglutarate (2OG)-dependent dioxygenases catalyze C-H activation while performing a wide range of chemical transformations. In contrast to their heme analogues, non-heme iron centers afford greater structural flexibility with important implications for their diverse catalytic mechanisms. We characterize an <i>in situ</i> structural model of the putative transient ferric intermediate of 2OG:taurine dioxygenase (TauD) by using a combination of spectroelectrochemical and semi-empirical computational methods, demonstrating that the Fe (III/II) transition involves a substantial, fully reversible, redox-linked conformational change at the active site. This rearrangement alters the apparent redox potential of the active site between -127 mV for reduction of the ferric state and 171 mV for oxidation of the ferrous state of the 2OG-Fe-TauD complex. Structural perturbations exhibit limited sensitivity to mediator concentrations and potential pulse duration. Similar changes were observed in the Fe-TauD and taurine-2OG-Fe-TauD complexes, thus attributing the reorganization to the protein moiety rather than the cosubstrates. Redox difference infrared spectra indicate a reorganization of the protein backbone in addition to the involvement of carboxylate and histidine ligands. Quantitative modeling of the transient redox response using two alternative reaction schemes across a variety of experimental conditions strongly supports the proposal for intrinsic protein reorganization as the origin of the experimental observations.

scholarly journals Time-Resolved Measurements and Master Equation Modelling of the Unimolecular Decomposition of CH3OCH2

2020 ◽  
Vol 234 (7-9) ◽  
pp. 1233-1250 ◽  
Author(s):  
Arrke J. Eskola ◽  
Mark A. Blitz ◽  
Michael J. Pilling ◽  
Paul W. Seakins ◽  
Robin J. Shannon
Keyword(s):  
Energy Transfer ◽  
Master Equation ◽  
Rate Coefficient ◽  
Zero Point ◽  
Buffer Gas ◽  
Unimolecular Decomposition ◽  
Time Resolved ◽  
Point Energy ◽  
Wide Range ◽  
Transfer Parameters

AbstractThe rate coefficient for the unimolecular decomposition of CH3OCH2, k1, has been measured in time-resolved experiments by monitoring the HCHO product. CH3OCH2 was rapidly and cleanly generated by 248 nm excimer photolysis of oxalyl chloride, (ClCO)2, in an excess of CH3OCH3, and an excimer pumped dye laser tuned to 353.16 nm was used to probe HCHO via laser induced fluorescence. k1(T,p) was measured over the ranges: 573–673 K and 0.1–4.3 × 1018 molecule cm−3 with a helium bath gas. In addition, some experiments were carried out with nitrogen as the bath gas. Ab initio calculations on CH3OCH2 decomposition were carried out and a transition-state for decomposition to CH3 and H2CO was identified. This information was used in a master equation rate calculation, using the MESMER code, where the zero-point-energy corrected barrier to reaction, ΔE0,1, and the energy transfer parameters, ⟨ΔEdown⟩ × Tn, were the adjusted parameters to best fit the experimental data, with helium as the buffer gas. The data were combined with earlier measurements by Loucks and Laidler (Can J. Chem.1967, 45, 2767), with dimethyl ether as the third body, reinterpreted using current literature for the rate coefficient for recombination of CH3OCH2. This analysis returned ΔE0,1 = (112.3 ± 0.6) kJ mol−1, and leads to $k_{1}^{\infty}(T)=2.9\times{10^{12}}$ (T/300)2.5 exp(−106.8 kJ mol−1/RT). Using this model, limited experiments with nitrogen as the bath gas allowed N2 energy transfer parameters to be identified and then further MESMER simulations were carried out, where N2 was the buffer gas, to generate k1(T,p) over a wide range of conditions: 300–1000 K and N2 = 1012–1025 molecule cm−3. The resulting k1(T,p) has been parameterized using a Troe-expression, so that they can be readily be incorporated into combustion models. In addition, k1(T,p) has been parametrized using PLOG for the buffer gases, He, CH3OCH3 and N2.

Detector commissioning and development for PETRA-III

2010 ◽  
Vol 1 (SRMS-7) ◽  
Author(s):  
David Pennicard ◽  
Heinz Graafsma ◽  
Michael Lohmann
Keyword(s):  
High Speed ◽  
Materials Science ◽  
Dynamic Range ◽  
Photon Counting ◽  
High Energy ◽  
X Rays ◽  
Silicon Detectors ◽  
Time Resolved ◽  
Wide Range ◽  
Synchrotron Light

The new synchrotron light source PETRA-III produced its first beam last year. The extremely high brilliance of PETRA-III and the large energy range of many of its beamlines make it useful for a wide range of experiments, particularly in materials science. The detectors at PETRA-III will need to meet several requirements, such as operation across a wide dynamic range, high-speed readout and good quantum efficiency even at high photon energies. PETRA-III beamlines with lower photon energies will typically be equipped with photon-counting silicon detectors for two-dimensional detection and silicon drift detectors for spectroscopy and higher-energy beamlines will use scintillators coupled to cameras or photomultiplier tubes. Longer-term developments include ‘high-Z’ semiconductors for detecting high-energy X-rays, photon-counting readout chips with smaller pixels and higher frame rates and pixellated avalanche photodiodes for time-resolved experiments.

scholarly journals Analysis of the HindIII-catalyzed reaction by time-resolved crystallography

2015 ◽  
Vol 71 (2) ◽  
pp. 256-265 ◽  
Author(s):  
Takashi Kawamura ◽  
Tomoki Kobayashi ◽  
Nobuhisa Watanabe
Keyword(s):  
Electron Density ◽  
Active Site ◽  
Dna Cleavage ◽  
Metal Ion ◽  
Manganese Ions ◽  
Metal Ion Binding ◽  
Time Resolved ◽  
Manganese Ion ◽  
Ion Binding Sites ◽  
Metal Ion Binding Sites

In order to investigate the mechanism of the reaction catalyzed by HindIII, structures of HindIII–DNA complexes with varying durations of soaking time in cryoprotectant buffer containing manganese ions were determined by the freeze-trap method. In the crystal structures of the complexes obtained after soaking for a longer duration, two manganese ions, indicated by relatively higher electron density, are clearly observed at the two metal ion-binding sites in the active site of HindIII. The increase in the electron density of the two metal-ion peaks followed distinct pathways with increasing soaking times, suggesting variation in the binding rate constant for the two metal sites. DNA cleavage is observed when the second manganese ion appears, suggesting that HindIII uses the two-metal-ion mechanism, or alternatively that its reactivity is enhanced by the binding of the second metal ion. In addition, conformational change in a loop near the active site accompanies the catalytic reaction.

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