12 results on '"Basu, Shibom"'
Search Results
2. An automated platform for structural analysis of membrane proteins through serial crystallography
- Author
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Healey, Robert D., Basu, Shibom, Humm, Anne-Sophie, Leyrat, Cedric, Cong, Xiaojing, Golebiowski, Jérôme, Dupeux, Florine, Pica, Andrea, Granier, Sébastien, and Márquez, José Antonio
- Published
- 2021
- Full Text
- View/download PDF
3. Structural dynamics and functional cooperativity of human NQO1 by ambient temperature serial crystallography and simulations.
- Author
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Grieco, Alice, Boneta, Sergio, Gavira, José A., Pey, Angel L., Basu, Shibom, Orlans, Julien, Sanctis, Daniele de, Medina, Milagros, and Martin‐Garcia, Jose Manuel
- Abstract
The human NQO1 (hNQO1) is a flavin adenine nucleotide (FAD)‐dependent oxidoreductase that catalyzes the two‐electron reduction of quinones to hydroquinones, being essential for the antioxidant defense system, stabilization of tumor suppressors, and activation of quinone‐based chemotherapeutics. Moreover, it is overexpressed in several tumors, which makes it an attractive cancer drug target. To decipher new structural insights into the flavin reductive half‐reaction of the catalytic mechanism of hNQO1, we have carried serial crystallography experiments at new ID29 beamline of the ESRF to determine, to the best of our knowledge, the first structure of the hNQO1 in complex with NADH. We have also performed molecular dynamics simulations of free hNQO1 and in complex with NADH. This is the first structural evidence that the hNQO1 functional cooperativity is driven by structural communication between the active sites through long‐range propagation of cooperative effects across the hNQO1 structure. Both structural results and MD simulations have supported that the binding of NADH significantly decreases protein dynamics and stabilizes hNQO1 especially at the dimer core and interface. Altogether, these results pave the way for future time‐resolved studies, both at x‐ray free‐electron lasers and synchrotrons, of the dynamics of hNQO1 upon binding to NADH as well as during the FAD cofactor reductive half‐reaction. This knowledge will allow us to reveal unprecedented structural information of the relevance of the dynamics during the catalytic function of hNQO1. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
4. Detection of a Geminate Photoproduct of Bovine Cytochrome c Oxidase by Time-Resolved Serial Femtosecond Crystallography.
- Author
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Ishigami, Izumi, Carbajo, Sergio, Zatsepin, Nadia, Hikita, Masahide, Conrad, Chelsie E., Nelson, Garrett, Coe, Jesse, Basu, Shibom, Grant, Thomas, Seaberg, Matthew H., Sierra, Raymond G., Hunter, Mark S., Fromme, Petra, Fromme, Raimund, Rousseau, Denis L., and Yeh, Syun-Ru
- Published
- 2023
- Full Text
- View/download PDF
5. ID23‐2: an automated and high‐performance microfocus beamline for macromolecular crystallography at the ESRF.
- Author
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Nanao, Max, Basu, Shibom, Zander, Ulrich, Giraud, Thierry, Surr, John, Guijarro, Matias, Lentini, Mario, Felisaz, Franck, Sinoir, Jeremy, Morawe, Christian, Vivo, Amparo, Beteva, Antonia, Oscarsson, Marcus, Caserotto, Hugo, Dobias, Fabien, Flot, David, Nurizzo, Didier, Gigmes, Jonathan, Foos, Nicolas, and Siebrecht, Ralf
- Subjects
- *
CRYSTALLOGRAPHY , *SYNCHROTRON radiation , *DATA reduction , *OPTICS , *DIFFRACTOMETERS - Abstract
ID23‐2 is a fixed‐energy (14.2 keV) microfocus beamline at the European Synchrotron Radiation Facility (ESRF) dedicated to macromolecular crystallography. The optics and sample environment have recently been redesigned and rebuilt to take full advantage of the upgrade of the ESRF to the fourth generation Extremely Brilliant Source (ESRF‐EBS). The upgraded beamline now makes use of two sets of compound refractive lenses and multilayer mirrors to obtain a highly intense (>1013 photons s−1) focused microbeam (minimum size 1.5 µm × 3 µm full width at half‐maximum). The sample environment now includes a FLEX‐HCD sample changer/storage system, as well as a state‐of‐the‐art MD3Up high‐precision multi‐axis diffractometer. Automatic data reduction and analysis are also provided for more advanced protocols such as synchrotron serial crystallographic experiments. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
6. Discovery and Mechanism of Action of Small Molecule Inhibitors of Ceramidases**.
- Author
-
Healey, Robert D., Saied, Essa M., Cong, Xiaojing, Karsai, Gergely, Gabellier, Ludovic, Saint‐Paul, Julie, Del Nero, Elise, Jeannot, Sylvain, Drapeau, Marion, Fontanel, Simon, Maurel, Damien, Basu, Shibom, Leyrat, Cedric, Golebiowski, Jérôme, Bossis, Guillaume, Bechara, Cherine, Hornemann, Thorsten, Arenz, Christoph, and Granier, Sebastien
- Subjects
SMALL molecules ,AMIDASES ,HUMAN physiology ,MASS spectrometry ,CERAMIDES ,DRUG development - Abstract
Sphingolipid metabolism is tightly controlled by enzymes to regulate essential processes in human physiology. The central metabolite is ceramide, a pro‐apoptotic lipid catabolized by ceramidase enzymes to produce pro‐proliferative sphingosine‐1‐phosphate. Alkaline ceramidases are transmembrane enzymes that recently attracted attention for drug development in fatty liver diseases. However, due to their hydrophobic nature, no specific small molecule inhibitors have been reported. We present the discovery and mechanism of action of the first drug‐like inhibitors of alkaline ceramidase 3 (ACER3). In particular, we chemically engineered novel fluorescent ceramide substrates enabling screening of large compound libraries and characterized enzyme:inhibitor interactions using mass spectrometry and MD simulations. In addition to revealing a new paradigm for inhibition of lipid metabolising enzymes with non‐lipidic small molecules, our data lay the ground for targeting ACER3 in drug discovery efforts. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
7. In situ serial crystallography facilitates 96-well plate structural analysis at low symmetry.
- Author
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Foos N, Florial JB, Eymery M, Sinoir J, Felisaz F, Oscarsson M, Beteva A, Bowler MW, Nurizzo D, Papp G, Soler-Lopez M, Nanao M, Basu S, and McCarthy AA
- Subjects
- Crystallography, X-Ray methods, Humans, Protein Conformation, Synchrotrons, Crystallization
- Abstract
The advent of serial crystallography has rejuvenated and popularized room-temperature X-ray crystal structure determination. Structures determined at physiological temperature reveal protein flexibility and dynamics. In addition, challenging samples (e.g. large complexes, membrane proteins and viruses) form fragile crystals that are often difficult to harvest for cryo-crystallography. Moreover, a typical serial crystallography experiment requires a large number of microcrystals, mainly achievable through batch crystallization. Many medically relevant samples are expressed in mammalian cell lines, producing a meager quantity of protein that is incompatible with batch crystallization. This can limit the scope of serial crystallography approaches. Direct in situ data collection from a 96-well crystallization plate enables not only the identification of the best diffracting crystallization condition but also the possibility for structure determination under ambient conditions. Here, we describe an in situ serial crystallography (iSX) approach, facilitating direct measurement from crystallization plates mounted on a rapidly exchangeable universal plate holder deployed at a microfocus beamline, ID23-2, at the European Synchrotron Radiation Facility. We applied our iSX approach on a challenging project, autotaxin, a therapeutic target expressed in a stable human cell line, to determine the structure in the lowest-symmetry P1 space group at 3.0 Å resolution. Our in situ data collection strategy provided a complete dataset for structure determination while screening various crystallization conditions. Our data analysis reveals that the iSX approach is highly efficient at a microfocus beamline, improving throughput and demonstrating how crystallization plates can be routinely used as an alternative method of presenting samples for serial crystallography experiments at synchrotrons., (open access.)
- Published
- 2024
- Full Text
- View/download PDF
8. Structural basis of tRNA recognition by the m 3 C RNA methyltransferase METTL6 in complex with SerRS seryl-tRNA synthetase.
- Author
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Throll P, G Dolce L, Rico-Lastres P, Arnold K, Tengo L, Basu S, Kaiser S, Schneider R, and Kowalinski E
- Abstract
Methylation of cytosine 32 in the anticodon loop of tRNAs to 3-methylcytosine (m
3 C) is crucial for cellular translation fidelity. Misregulation of the RNA methyltransferases setting this modification can cause aggressive cancers and metabolic disturbances. Here, we report the cryo-electron microscopy structure of the human m3 C tRNA methyltransferase METTL6 in complex with seryl-tRNA synthetase (SerRS) and their common substrate tRNASer . Through the complex structure, we identify the tRNA-binding domain of METTL6. We show that SerRS acts as the tRNASer substrate selection factor for METTL6. We demonstrate that SerRS augments the methylation activity of METTL6 and that direct contacts between METTL6 and SerRS are necessary for efficient tRNASer methylation. Finally, on the basis of the structure of METTL6 in complex with SerRS and tRNASer , we postulate a universal tRNA-binding mode for m3 C RNA methyltransferases, including METTL2 and METTL8, suggesting that these mammalian paralogs use similar ways to engage their respective tRNA substrates and cofactors., (© 2024. The Author(s).)- Published
- 2024
- Full Text
- View/download PDF
9. Structural dynamics and functional cooperativity of human NQO1 by ambient temperature serial crystallography and simulations.
- Author
-
Grieco A, Boneta S, Gavira JA, Pey AL, Basu S, Orlans J, Sanctis D, Medina M, and Martin-Garcia JM
- Subjects
- Humans, Crystallography, Temperature, NAD, Flavins, Crystallography, X-Ray, NAD(P)H Dehydrogenase (Quinone), Antineoplastic Agents chemistry, Neoplasms
- Abstract
The human NQO1 (hNQO1) is a flavin adenine nucleotide (FAD)-dependent oxidoreductase that catalyzes the two-electron reduction of quinones to hydroquinones, being essential for the antioxidant defense system, stabilization of tumor suppressors, and activation of quinone-based chemotherapeutics. Moreover, it is overexpressed in several tumors, which makes it an attractive cancer drug target. To decipher new structural insights into the flavin reductive half-reaction of the catalytic mechanism of hNQO1, we have carried serial crystallography experiments at new ID29 beamline of the ESRF to determine, to the best of our knowledge, the first structure of the hNQO1 in complex with NADH. We have also performed molecular dynamics simulations of free hNQO1 and in complex with NADH. This is the first structural evidence that the hNQO1 functional cooperativity is driven by structural communication between the active sites through long-range propagation of cooperative effects across the hNQO1 structure. Both structural results and MD simulations have supported that the binding of NADH significantly decreases protein dynamics and stabilizes hNQO1 especially at the dimer core and interface. Altogether, these results pave the way for future time-resolved studies, both at x-ray free-electron lasers and synchrotrons, of the dynamics of hNQO1 upon binding to NADH as well as during the FAD cofactor reductive half-reaction. This knowledge will allow us to reveal unprecedented structural information of the relevance of the dynamics during the catalytic function of hNQO1., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)
- Published
- 2024
- Full Text
- View/download PDF
10. Droplet microfluidics for time-resolved serial crystallography.
- Author
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Stubbs J, Hornsey T, Hanrahan N, Esteban LB, Bolton R, Malý M, Basu S, Orlans J, de Sanctis D, Shim JU, Shaw Stewart PD, Orville AM, Tews I, and West J
- Subjects
- Crystallography, Cognition, Convection, Microfluidics, Arabidopsis
- Abstract
Serial crystallography requires large numbers of microcrystals and robust strategies to rapidly apply substrates to initiate reactions in time-resolved studies. Here, we report the use of droplet miniaturization for the controlled production of uniform crystals, providing an avenue for controlled substrate addition and synchronous reaction initiation. The approach was evaluated using two enzymatic systems, yielding 3 µm crystals of lysozyme and 2 µm crystals of Pdx1, an Arabidopsis enzyme involved in vitamin B6 biosynthesis. A seeding strategy was used to overcome the improbability of Pdx1 nucleation occurring with diminishing droplet volumes. Convection within droplets was exploited for rapid crystal mixing with ligands. Mixing times of <2 ms were achieved. Droplet microfluidics for crystal size engineering and rapid micromixing can be utilized to advance time-resolved serial crystallography., (open access.)
- Published
- 2024
- Full Text
- View/download PDF
11. Detection of a geminate photoproduct of bovine cytochrome c oxidase by time-resolved serial femtosecond crystallography.
- Author
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Ishigami I, Carbajo S, Zatsepin N, Hikita M, Conrad CE, Nelson G, Coe J, Basu S, Grant T, Seaberg MH, Sierra RG, Hunter MS, Fromme P, Fromme R, Rousseau DL, and Yeh SR
- Abstract
Cytochrome c oxidase (C c O) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a heme b group in their active sites, C c O has a unique binuclear center (BNC) comprised of a copper atom (Cu
B ) and a heme a3 iron, where O2 binds and is reduced to water. CO is a versatile O2 surrogate in ligand binding and escape reactions. Previous time-resolved spectroscopic studies of the CO complexes of bovine C c O (bC c O) revealed that photolyzing CO from the heme a3 iron leads to a metastable intermediate (CuB -CO), where CO is bound to CuB , before it escapes out of the BNC. Here, with a time-resolved serial femtosecond X-ray crystallography-based pump-probe method, we detected a geminate photoproduct of the bC c O-CO complex, where CO is dissociated from the heme a3 iron and moved to a temporary binding site midway between the CuB and the heme a3 iron, while the locations of the two metal centers and the conformation of the Helix-X, housing the proximal histidine ligand of the heme a3 iron, remain in the CO complex state. This new structure, combined with other reported structures of bC c O, allows the full definition of the ligand dissociation trajectory, as well as the associated protein dynamics.- Published
- 2023
- Full Text
- View/download PDF
12. Discovery and Mechanism of Action of Small Molecule Inhibitors of Ceramidases.
- Author
-
Healey RD, Saied EM, Cong X, Karsai G, Gabellier L, Saint-Paul J, Del Nero E, Jeannot S, Drapeau M, Fontanel S, Maurel D, Basu S, Leyrat C, Golebiowski J, Bossis G, Bechara C, Hornemann T, Arenz C, and Granier S
- Subjects
- Humans, Alkaline Ceramidase metabolism, Alkaline Ceramidase antagonists & inhibitors, Molecular Dynamics Simulation, Molecular Structure, Ceramides metabolism, Ceramides chemistry, Ceramidases antagonists & inhibitors, Ceramidases metabolism, Small Molecule Libraries chemistry, Small Molecule Libraries pharmacology, Small Molecule Libraries metabolism, Enzyme Inhibitors chemistry, Enzyme Inhibitors pharmacology, Enzyme Inhibitors metabolism, Drug Discovery
- Abstract
Sphingolipid metabolism is tightly controlled by enzymes to regulate essential processes in human physiology. The central metabolite is ceramide, a pro-apoptotic lipid catabolized by ceramidase enzymes to produce pro-proliferative sphingosine-1-phosphate. Alkaline ceramidases are transmembrane enzymes that recently attracted attention for drug development in fatty liver diseases. However, due to their hydrophobic nature, no specific small molecule inhibitors have been reported. We present the discovery and mechanism of action of the first drug-like inhibitors of alkaline ceramidase 3 (ACER3). In particular, we chemically engineered novel fluorescent ceramide substrates enabling screening of large compound libraries and characterized enzyme:inhibitor interactions using mass spectrometry and MD simulations. In addition to revealing a new paradigm for inhibition of lipid metabolising enzymes with non-lipidic small molecules, our data lay the ground for targeting ACER3 in drug discovery efforts., (© 2021 Wiley-VCH GmbH.)
- Published
- 2022
- Full Text
- View/download PDF
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