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1. Stiffness heterogeneity of small viral capsids

Author

Menou, Lucas, Carrasco-Salas, Yeraldinne, Lecoq, Lauriane, Salvetti, Anna, Faivre-Moskalenko, Cendrine, and Castelnovo, Martin

Subjects
Condensed Matter - Soft Condensed Matter
Abstract

Nanoindentation of viral capsids provides an efficient tool in order to probe their elastic properties. We investigate in the present work the various sources of stiffness heterogeneity as observed in Atomic Force Microscopy (AFM) experiments. By combining experimental results with both numerical and analytical modeling, we first show that for small viruses a position-dependent stiffness is observed. This effect is strong and has not been properly taken into account previously. Moreover, we show that a geometrical model is able to reproduce this effect quantitatively. Our work suggests alternative ways of measuring stiffness heterogeneities on small viral capsids. This is illustrated on two different viral capsids: Adeno Associated Virus serotype 8 (AAV8) and Hepatitis B Virus (HBV with T=4). We discuss our results in the light of continuous elasticity modeling., Comment: Physical Review E , American Physical Society (APS), In press

Published
2021
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6. Biomolecular solid-state NMR spectroscopy at 1200 MHz: the gain in resolution

Author

Callon, Morgane, Malär, Alexander A., Pfister, Sara, Římal, Václav, Weber, Marco E., Wiegand, Thomas, Zehnder, Johannes, Chávez, Matías, Cadalbert, Riccardo, Deb, Rajdeep, Däpp, Alexander, Fogeron, Marie-Laure, Hunkeler, Andreas, Lecoq, Lauriane, Torosyan, Anahit, Zyla, Dawid, Glockshuber, Rudolf, Jonas, Stefanie, Nassal, Michael, Ernst, Matthias, Böckmann, Anja, and Meier, Beat H.

Published
2021
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7. Analysis of the structure and interactions of the SARS-CoV-2 ORF7b accessory protein.

Author

Minh-Ha Nguyen, Palfy, Gyula, Fogeron, Marie-Laure, Pedrosa, Martí Ninot, Zehnder, Johannes, Rimal, Vaclav, Callon, Morgane, Lecoq, Lauriane, Barnes, Alexander, Meier, Beat H., and Böckmann, Anja

Subjects
LEUCINE zippers, MEMBRANE proteins, TRANSMEMBRANE domains, PHOSPHOLAMBAN, IMMUNOREGULATION
Abstract

SARS-CoV- 2 carries a sizeable number of proteins that are accessory to replication but may be essential for virus-host interactions and modulation of the host immune response. Here, we investigated the structure and interactions of the largely unknown ORF7b, a small membranous accessory membrane protein of SARS-CoV- 2. We show that structural predictions indicate a transmembrane (TM) leucine zipper for ORF7b, and experimentally confirm the predominantly a-helical secondary structure within a phospholipid membrane mimetic by solid-state NMR. We also show that ORF7b forms heterogeneous higher-order multimers. We determined ORF7b interactions with cellular TM leucine zipper proteins using both biochemical and NMR approaches, providing evidence for ORF7b interaction with the TM domains of E-cadherin, as well as phospholamban. Our results place ORF7b as a hypothetical interferer in cellular processes that utilize leucine zipper motifs in transmembrane multimerization domains. [ABSTRACT FROM AUTHOR]

Published
2024
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8. Synthetic injectable and porous hydrogels for the formation of skeletal muscle fibers: Novel perspectives for the acellular repair of substantial volumetric muscle loss.

Author

Griveau, Louise, Bouvet, Marion, Christin, Emilie, Paret, Cloé, Lecoq, Lauriane, Radix, Sylvie, Laumonier, Thomas, Sohier, Jerome, and Gache, Vincent

Subjects
SKELETAL muscle injuries, MUSCLE cells, STEM cells, CONNECTIVE tissues, TIBIALIS anterior, GUIDED tissue regeneration
Abstract

In severe skeletal muscle damage, muscle tissue regeneration process has to face the loss of resident muscle stem cells (MuSCs) and the lack of connective tissue necessary to guide the regeneration process. Biocompatible and standardized 3D structures that can be injected to the muscle injury site, conforming to the defect shape while actively guiding the repair process, holds great promise for skeletal muscle tissue regeneration. In this study, we explore the use of an injectable and porous lysine dendrimer/polyethylene glycol (DGL/PEG) hydrogel as an acellular support for skeletal muscle regeneration. We adjusted the DGL/PEG composition to achieve a stiffness conducive to the attachment and proliferation of murine immortalized myoblasts and human primary muscle stems cells, sustaining the formation and maturation of muscle fibers in vitro. We then evaluated the potential of one selected "myogenic-porous hydrogel" as a supportive structure for muscle repair in a large tibialis anterior muscle defect in rats. This injectable and porous formulation filled the defect, promoting rapid cellularization with the presence of endothelial cells, macrophages, and myoblasts, thereby supporting neo-myogenesis more specifically at the interface between the wound edges and the hydrogel. The selected porous DGL/PEG hydrogel acted as a guiding scaffold at the periphery of the defect, facilitating the formation and anchorage of aligned muscle fibers 21 days after injury. Overall, our results indicate DGL/PEG porous injectable hydrogel potential to create a pro-regenerative environment for muscle cells after large skeletal muscle injuries, paving the way for acellular treatment in regenerative muscle medicine. [ABSTRACT FROM AUTHOR]

Published
2024
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12. Comprehensive Fragment Screening of the SARS‐CoV‐2 Proteome Explores Novel Chemical Space for Drug Development

Author

Berg, Hannes, Wirtz Martin, Maria A., Altincekic, Nadide, Alshamleh, Islam, Kaur Bains, Jasleen, Blechar, Julius, Ceylan, Betül, Jesus, Vanessa de, Dhamotharan, Karthikeyan, Fuks, Christin, Gande, Santosh L., Hargittay, Bruno, Hohmann, Katharina F., Hutchison, Marie T., Korn, Sophie Marianne, Krishnathas, Robin, Kutz, Felicitas, Linhard, Verena, Matzel, Tobias, Meiser, Nathalie, Niesteruk, Anna, Pyper, Dennis J., Schulte, Linda, Trucks, Sven, Azzaoui, Kamal, Blommers, Marcel J. J., Gadiya, Yojana, Karki, Reagon, Zaliani, Andrea, Gribbon, Philip, Silva Almeida, Marcius da, Dinis Anobom, Cristiane, Bula, Anna L., Bütikofer, Matthias, Putinhon Caruso, Ícaro, Caterina Felli, Isabella, Da Poian, Andrea T., Cardoso de Amorim, Gisele, Fourkiotis, Nikolaos K., Gallo, Angelo, Ghosh, Dhiman, Gomes‐Neto, Francisco, Gorbatyuk, Oksana, Hao, Bing, Kurauskas, Vilius, Lecoq, Lauriane, Li, Yunfeng, Cunha Mebus‐Antunes, Nathane, Mompeán, Miguel, Cristtina Neves‐Martins, Thais, Ninot‐Pedrosa, Martí, Pinheiro, Anderson S., Pontoriero, Letizia, Pustovalova, Yulia, Riek, Roland, Robertson, Angus J., Jose Abi Saad, Marie, Treviño, Miguel Á., Tsika, Aikaterini C., Almeida, Fabio C. L., Bax, Ad, Henzler‐Wildman, Katherine, Hoch, Jeffrey C., Jaudzems, Kristaps, Laurents, Douglas V., Orts, Julien, Pierattelli, Roberta, Spyroulias, Georgios A., Duchardt‐Ferner, Elke, Ferner, Jan, Fürtig, Boris, Hengesbach, Martin, Löhr, Frank, Qureshi, Nusrat, Richter, Christian, Saxena, Krishna, Schlundt, Andreas, Sreeramulu, Sridhar, Wacker, Anna, Weigand, Julia E., Wirmer‐Bartoschek, Julia, Wöhnert, Jens, Schwalbe, Harald, Berg, Hannes, Wirtz Martin, Maria A., Altincekic, Nadide, Alshamleh, Islam, Kaur Bains, Jasleen, Blechar, Julius, Ceylan, Betül, Jesus, Vanessa de, Dhamotharan, Karthikeyan, Fuks, Christin, Gande, Santosh L., Hargittay, Bruno, Hohmann, Katharina F., Hutchison, Marie T., Korn, Sophie Marianne, Krishnathas, Robin, Kutz, Felicitas, Linhard, Verena, Matzel, Tobias, Meiser, Nathalie, Niesteruk, Anna, Pyper, Dennis J., Schulte, Linda, Trucks, Sven, Azzaoui, Kamal, Blommers, Marcel J. J., Gadiya, Yojana, Karki, Reagon, Zaliani, Andrea, Gribbon, Philip, Silva Almeida, Marcius da, Dinis Anobom, Cristiane, Bula, Anna L., Bütikofer, Matthias, Putinhon Caruso, Ícaro, Caterina Felli, Isabella, Da Poian, Andrea T., Cardoso de Amorim, Gisele, Fourkiotis, Nikolaos K., Gallo, Angelo, Ghosh, Dhiman, Gomes‐Neto, Francisco, Gorbatyuk, Oksana, Hao, Bing, Kurauskas, Vilius, Lecoq, Lauriane, Li, Yunfeng, Cunha Mebus‐Antunes, Nathane, Mompeán, Miguel, Cristtina Neves‐Martins, Thais, Ninot‐Pedrosa, Martí, Pinheiro, Anderson S., Pontoriero, Letizia, Pustovalova, Yulia, Riek, Roland, Robertson, Angus J., Jose Abi Saad, Marie, Treviño, Miguel Á., Tsika, Aikaterini C., Almeida, Fabio C. L., Bax, Ad, Henzler‐Wildman, Katherine, Hoch, Jeffrey C., Jaudzems, Kristaps, Laurents, Douglas V., Orts, Julien, Pierattelli, Roberta, Spyroulias, Georgios A., Duchardt‐Ferner, Elke, Ferner, Jan, Fürtig, Boris, Hengesbach, Martin, Löhr, Frank, Qureshi, Nusrat, Richter, Christian, Saxena, Krishna, Schlundt, Andreas, Sreeramulu, Sridhar, Wacker, Anna, Weigand, Julia E., Wirmer‐Bartoschek, Julia, Wöhnert, Jens, and Schwalbe, Harald

Abstract

SARS‐CoV‐2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti‐virals. Within the international Covid19‐NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR‐detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure‐based drug design against the SCoV2 proteome.

Published
2024

14. NMR side-chain assignments of the Crimean–Congo hemorrhagic fever virus glycoprotein n cytosolic domain.

Author

Brigandat, Louis, Laux, Maëlys, Marteau, Caroline, Cole, Laura, Böckmann, Anja, Lecoq, Lauriane, Fogeron, Marie-Laure, and Callon, Morgane

Subjects
HEMORRHAGIC fever, ZINC-finger proteins, ZINC ions, PROTEIN synthesis, AMINO acids
Abstract

We assigned the side-chain resonances of the Crimean–Congo hemorrhagic fever virus (CCHFV) glycoprotein n (Gn) cytosolic domain that is 69 amino acids long to complete the backbone resonances previously published by Estrada et al. (2011). The process was facilitated by three factors. First, sample preparation using cell-free protein synthesis (CFPS) was completed in less than 2 d and allowed for correct zinc finger formation by adding zinc ions to the reaction. Second, access to NMR platforms with standardized pulse sequences allowed for data acquisition in 18 d. Third, data analysis using the online platform NMRtist allowed sequential resonance assignments to be made in a day, and assignments were verified and finalized in less than a week. Our work thus provides an example of how NMR assignments, including side chains, of small and well-behaved proteins can be approached in a rapid routine, at protein concentrations of 150 µ M. [ABSTRACT FROM AUTHOR]

Published
2024
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21. Comprehensive Fragment Screening of the SARS‐CoV‐2 Proteome Explores Novel Chemical Space for Drug Development

Author

Berg, Hannes, primary, Wirtz Martin, Maria A., additional, Altincekic, Nadide, additional, Alshamleh, Islam, additional, Kaur Bains, Jasleen, additional, Blechar, Julius, additional, Ceylan, Betül, additional, de Jesus, Vanessa, additional, Dhamotharan, Karthikeyan, additional, Fuks, Christin, additional, Gande, Santosh L., additional, Hargittay, Bruno, additional, Hohmann, Katharina F., additional, Hutchison, Marie T., additional, Marianne Korn, Sophie, additional, Krishnathas, Robin, additional, Kutz, Felicitas, additional, Linhard, Verena, additional, Matzel, Tobias, additional, Meiser, Nathalie, additional, Niesteruk, Anna, additional, Pyper, Dennis J., additional, Schulte, Linda, additional, Trucks, Sven, additional, Azzaoui, Kamal, additional, Blommers, Marcel J. J., additional, Gadiya, Yojana, additional, Karki, Reagon, additional, Zaliani, Andrea, additional, Gribbon, Philip, additional, da Silva Almeida, Marcius, additional, Dinis Anobom, Cristiane, additional, Bula, Anna L., additional, Bütikofer, Matthias, additional, Putinhon Caruso, Ícaro, additional, Caterina Felli, Isabella, additional, Da Poian, Andrea T., additional, Cardoso de Amorim, Gisele, additional, Fourkiotis, Nikolaos K., additional, Gallo, Angelo, additional, Ghosh, Dhiman, additional, Gomes‐Neto, Francisco, additional, Gorbatyuk, Oksana, additional, Hao, Bing, additional, Kurauskas, Vilius, additional, Lecoq, Lauriane, additional, Li, Yunfeng, additional, Cunha Mebus‐Antunes, Nathane, additional, Mompeán, Miguel, additional, Cristtina Neves‐Martins, Thais, additional, Ninot‐Pedrosa, Martí, additional, Pinheiro, Anderson S., additional, Pontoriero, Letizia, additional, Pustovalova, Yulia, additional, Riek, Roland, additional, Robertson, Angus J., additional, Jose Abi Saad, Marie, additional, Treviño, Miguel Á., additional, Tsika, Aikaterini C., additional, Almeida, Fabio C. L., additional, Bax, Ad, additional, Henzler‐Wildman, Katherine, additional, Hoch, Jeffrey C., additional, Jaudzems, Kristaps, additional, Laurents, Douglas V., additional, Orts, Julien, additional, Pierattelli, Roberta, additional, Spyroulias, Georgios A., additional, Duchardt‐Ferner, Elke, additional, Ferner, Jan, additional, Fürtig, Boris, additional, Hengesbach, Martin, additional, Löhr, Frank, additional, Qureshi, Nusrat, additional, Richter, Christian, additional, Saxena, Krishna, additional, Schlundt, Andreas, additional, Sreeramulu, Sridhar, additional, Wacker, Anna, additional, Weigand, Julia E., additional, Wirmer‐Bartoschek, Julia, additional, Wöhnert, Jens, additional, and Schwalbe, Harald, additional

Published
2022
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22. Molecular elucidation of drug-induced abnormal assemblies of the Hepatitis B Virus capsid protein by solid-state NMR

Author

Lecoq, Lauriane, primary, Brigandat, Louis, additional, Huber, Rebecca, additional, Fogeron, Marie-Laure, additional, Callon, Morgane, additional, Malär, Alexander, additional, Wang, Shishan, additional, Dujardin, Marie, additional, Briday, Mathilde, additional, Wiegand, Thomas, additional, Durantel, David, additional, Burdette, Dara, additional, Berke, Jan Martin, additional, Meier, Beat H., additional, Nassal, Michael, additional, and Böckmann, Anja, additional

Published
2022
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25. Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

Author

Altincekic, Nadide, Korn, Sophie Marianne, Qureshi, Nusrat Shahin, Dujardin, Marie, Ninot-Pedrosa, Martí, Abele, Rupert, Abi Saad, Marie Jose, Alfano, Caterina, Almeida, Fabio C. L., Alshamleh, Islam, de Amorim, Gisele Cardoso, Anderson, Thomas K., Anobom, Cristiane D., Anorma, Chelsea, Bains, Jasleen Kaur, Bax, Adriaan, Blackledge, Martin, Blechar, Julius, Böckmann, Anja, Brigandat, Louis, Bula, Anna, Bütikofer, Matthias, Camacho-Zarco, Aldo R., Carlomagno, Teresa, Caruso, Icaro Putinhon, Ceylan, Betül, Chaikuad, Apirat, Chu, Feixia, Cole, Laura, Crosby, Marquise G., Jesus, Vanessa de, Dhamotharan, Karthikeyan, Felli, Isabella C., Ferner, Jan, Fleischmann, Yanick, Fogeron, Marie-Laure, Fourkiotis, Nikolaos K., Fuks, Christin, Fürtig, Boris, Gallo, Angelo, Gande, Santosh L., Gerez, Juan Atilio, Ghosh, Dhiman, Gomes-Neto, Francisco, Gorbatyuk, Oksana, Guseva, Serafima, Hacker, Carolin, Häfner, Sabine, Hao, Bing, Hargittay, Bruno, Henzler-Wildman, K., Hoch, Jeffrey C., Hohmann, Katharina F., Hutchison, Marie T., Jaudzems, Kristaps, Jović, Katarina, Kaderli, Janina, Kalniņš, Gints, Kaņepe, Iveta, Kirchdoerfer, Robert N., Kirkpatrick, John, Knapp, Stefan, Krishnathas, Robin, Kutz, Felicitas, Lage, Susanne zur, Lambertz, Roderick, Lang, Andras, Laurents, Douglas, Lecoq, Lauriane, Linhard, Verena, Löhr, Frank, Malki, Anas, Bessa, Luiza Mamigonian, Martin, Rachel W., Matzel, Tobias, Maurin, Damien, McNutt, Seth W., Mebus-Antunes, Nathane Cunha, Meier, Beat H., Meiser, Nathalie, Mompeán, Miguel, Monaca, Elisa, Montserret, Roland, Mariño Perez, Laura, Moser, Celine, Muhle-Goll, Claudia, Neves-Martins, Thais Cristtina, Ni, Xiamonin, Norton-Baker, Brenna, Pierattelli, Roberta, Pontoriero, Letizia, Pustovalova, Yulia, Ohlenschläger, Oliver, Orts, Julien, Da Poian, Andrea T., Pyper, Dennis J., Richter, Christian, Riek, Roland, Rienstra, Chad M., Robertson, Angus, Pinheiro, Anderson S., Sabbatella, Raffaele, Salvi, Nicola, Saxena, Krishna, Schulte, Linda, Schiavina, Marco, Schwalbe, Harald, Silber, Mara, da Silva Almeida, Marcius, Sprague-Piercy, Marc A., Spyroulias, Georgios A., Sreeramulu, Sridhar, Tants, Jan-Niklas, Tārs, Kaspars, Torres, Felix, Töws, Sabrina, Treviño, Miguel Á., Trucks, Sven, Tsika, Aikaterini C., Varga, Krisztina, Wang, Ying, Weber, Marco E., Weigand, Julia E., Wiedemann, Christoph, Wirmer-Bartoschek, Julia, Wirtz Martin, Maria Alexandra, Zehnder, Johannes, Hengesbach, Martin, Schlundt, Andreas, Altincekic, Nadide, Korn, Sophie Marianne, Qureshi, Nusrat Shahin, Dujardin, Marie, Ninot-Pedrosa, Martí, Abele, Rupert, Abi Saad, Marie Jose, Alfano, Caterina, Almeida, Fabio C. L., Alshamleh, Islam, de Amorim, Gisele Cardoso, Anderson, Thomas K., Anobom, Cristiane D., Anorma, Chelsea, Bains, Jasleen Kaur, Bax, Adriaan, Blackledge, Martin, Blechar, Julius, Böckmann, Anja, Brigandat, Louis, Bula, Anna, Bütikofer, Matthias, Camacho-Zarco, Aldo R., Carlomagno, Teresa, Caruso, Icaro Putinhon, Ceylan, Betül, Chaikuad, Apirat, Chu, Feixia, Cole, Laura, Crosby, Marquise G., Jesus, Vanessa de, Dhamotharan, Karthikeyan, Felli, Isabella C., Ferner, Jan, Fleischmann, Yanick, Fogeron, Marie-Laure, Fourkiotis, Nikolaos K., Fuks, Christin, Fürtig, Boris, Gallo, Angelo, Gande, Santosh L., Gerez, Juan Atilio, Ghosh, Dhiman, Gomes-Neto, Francisco, Gorbatyuk, Oksana, Guseva, Serafima, Hacker, Carolin, Häfner, Sabine, Hao, Bing, Hargittay, Bruno, Henzler-Wildman, K., Hoch, Jeffrey C., Hohmann, Katharina F., Hutchison, Marie T., Jaudzems, Kristaps, Jović, Katarina, Kaderli, Janina, Kalniņš, Gints, Kaņepe, Iveta, Kirchdoerfer, Robert N., Kirkpatrick, John, Knapp, Stefan, Krishnathas, Robin, Kutz, Felicitas, Lage, Susanne zur, Lambertz, Roderick, Lang, Andras, Laurents, Douglas, Lecoq, Lauriane, Linhard, Verena, Löhr, Frank, Malki, Anas, Bessa, Luiza Mamigonian, Martin, Rachel W., Matzel, Tobias, Maurin, Damien, McNutt, Seth W., Mebus-Antunes, Nathane Cunha, Meier, Beat H., Meiser, Nathalie, Mompeán, Miguel, Monaca, Elisa, Montserret, Roland, Mariño Perez, Laura, Moser, Celine, Muhle-Goll, Claudia, Neves-Martins, Thais Cristtina, Ni, Xiamonin, Norton-Baker, Brenna, Pierattelli, Roberta, Pontoriero, Letizia, Pustovalova, Yulia, Ohlenschläger, Oliver, Orts, Julien, Da Poian, Andrea T., Pyper, Dennis J., Richter, Christian, Riek, Roland, Rienstra, Chad M., Robertson, Angus, Pinheiro, Anderson S., Sabbatella, Raffaele, Salvi, Nicola, Saxena, Krishna, Schulte, Linda, Schiavina, Marco, Schwalbe, Harald, Silber, Mara, da Silva Almeida, Marcius, Sprague-Piercy, Marc A., Spyroulias, Georgios A., Sreeramulu, Sridhar, Tants, Jan-Niklas, Tārs, Kaspars, Torres, Felix, Töws, Sabrina, Treviño, Miguel Á., Trucks, Sven, Tsika, Aikaterini C., Varga, Krisztina, Wang, Ying, Weber, Marco E., Weigand, Julia E., Wiedemann, Christoph, Wirmer-Bartoschek, Julia, Wirtz Martin, Maria Alexandra, Zehnder, Johannes, Hengesbach, Martin, and Schlundt, Andreas

Abstract

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.

Published
2022

26. Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development

Author

State of Hesse, German Research Foundation, European Commission, Ministero dell'Istruzione, dell'Università e della Ricerca, Agence Nationale de la Recherche (France), Centre National de la Recherche Scientifique (France), National Institutes of Health (US), National Science Foundation (US), Latvian Council of Science, Berg, Hannes [0000-0002-2060-4296], Wirtz Martin, Maria A. [0000-0002-0318-7785], Altincekic, Nadide [0000-0001-6370-3414], Alshamleh, Islam [0000-0001-6714-3602], Dhamotharan, Karthikeyan [0000-0003-0226-7350], Marianne Korn, Sophie [0000-0003-3798-3277], Schulte, Linda [0000-0002-9334-8908], da Silva Almeida, Marcius [0000-0003-4921-8185], Caterina Felli, Isabella [0000-0002-6018-9090], Fourkiotis, Nikolaos K. [0000-0002-5197-4142], Gallo, Angelo [0000-0001-9778-4822], Ninot-Pedrosa, Martí [0000-0003-2851-9990], Pontoriero, Letizia [0000-0002-5586-1305], Treviño, Miguel A. [0000-0002-0738-5973], Tsika, Aikaterini C. [000-0002-3723-0606], Almeida, Fabio C.L. [0000-0001-6046-7006], Bax, Ad [0000-0002-9809-5700], Henzler-Wildman, Katherine [0000-0002-5295-2121], Hoch, Jeffrey C. [0000-0002-9230-2019], Jaudzems, Kristaps [0000-0003-3922-2447], Laurents, D.V. [0000-0002-4187-165X], Ferner, Jan [0000-0002-2009-3203], Hengesbach, Martin [0000-0001-9414-1602], Löhr, Frank [0000-0001-6399-9497], Qureshi, Nusrat [0000-0002-5753-5984], Richter, Christian [0000-0002-5420-2826], Schlundt, Andreas [0000-0003-2254-7560], Weigand, Julia E. [0000-0003-4247-1348], Wirmer-Bartoschek, Julia [0000-0002-0642-1311], Schwalbe, Harald [0000-0001-5693-7909], Berg, Hannes, Wirtz Martin, Maria A., Altincekic, Nadide, Alshamleh, Islam, Kaur Bains, Jasleen, Blechar, Julius, Ceylan, Betül, Jesus, Vanessa de, Dhamotharan, Karthikeyan, Fuks, Christin, Gande, Santosh L., Hargittay, Bruno, Hohmann, Katharina F., Hutchison, Marie T., Marianne Korn, Sophie, Krishnathas, Robin, Kutz, Felicitas, Linhard, Verena, Matzel, Tobias, Meiser, Nathalie, Niesteruk, Anna, Pyper, Dennis J., Schulte, Linda, Trucks, Sven, Azzaoui, Kamal, Blommers, Marcel J.J., Gadiya, Yojana, Karki, Reagon, Zaliani, Andrea, Gribbon, Philip, da Silva Almeida, Marcius, Dinis Anobom, Cristiane, Bula, Anna L., Bütikofer, Matthias, Putinhon Caruso, Ícaro, Caterina Felli, Isabella, Da Poian, Andrea T., Cardoso de Amorim, Gisele, Fourkiotis, Nikolaos K., Gallo, Angelo, Ghosh, Dhiman, Gomes-Neto, Francisco, Gorbatyuk, Oksana, Hao, Bing, Kurauskas, Vilius, Lecoq, Lauriane, Li, Yunfeng, Cunha Mebus-Antunes, Nathane, Mompeán, Miguel, Cristtina Neves-Martins, Thais, Ninot-Pedrosa, Martí, Pinheiro, Anderson S.., Pontoriero, Letizia, Pustovalova, Yulia, Riek, Roland, Robertson, Angus J., Jose Abi Saad, Marie, Treviño, Miguel A., Tsika, Aikaterini C., Almeida, Fabio C.L., Bax, Ad, Henzler-Wildman, Katherine, Hoch, Jeffrey C., Jaudzems, Kristaps, Laurents, Douglas V., Orts, Julien, Pierattelli, Roberta, Spyroulias, Georgios A., Duchardt-Ferner, Elke, Ferner, Jan, Fürtig, Boris, Hengesbach, Martin, Löhr, Frank, Qureshi, Nusrat, Richter, Christian, Saxena, Krishna, Schlundt, Andreas, Sreeramulu, Sridhar, Wacker, Anna, Weigand, Julia E., Wirmer-Bartoschek, Julia, Wöhnert, Jens, Schwalbe, Harald, State of Hesse, German Research Foundation, European Commission, Ministero dell'Istruzione, dell'Università e della Ricerca, Agence Nationale de la Recherche (France), Centre National de la Recherche Scientifique (France), National Institutes of Health (US), National Science Foundation (US), Latvian Council of Science, Berg, Hannes [0000-0002-2060-4296], Wirtz Martin, Maria A. [0000-0002-0318-7785], Altincekic, Nadide [0000-0001-6370-3414], Alshamleh, Islam [0000-0001-6714-3602], Dhamotharan, Karthikeyan [0000-0003-0226-7350], Marianne Korn, Sophie [0000-0003-3798-3277], Schulte, Linda [0000-0002-9334-8908], da Silva Almeida, Marcius [0000-0003-4921-8185], Caterina Felli, Isabella [0000-0002-6018-9090], Fourkiotis, Nikolaos K. [0000-0002-5197-4142], Gallo, Angelo [0000-0001-9778-4822], Ninot-Pedrosa, Martí [0000-0003-2851-9990], Pontoriero, Letizia [0000-0002-5586-1305], Treviño, Miguel A. [0000-0002-0738-5973], Tsika, Aikaterini C. [000-0002-3723-0606], Almeida, Fabio C.L. [0000-0001-6046-7006], Bax, Ad [0000-0002-9809-5700], Henzler-Wildman, Katherine [0000-0002-5295-2121], Hoch, Jeffrey C. [0000-0002-9230-2019], Jaudzems, Kristaps [0000-0003-3922-2447], Laurents, D.V. [0000-0002-4187-165X], Ferner, Jan [0000-0002-2009-3203], Hengesbach, Martin [0000-0001-9414-1602], Löhr, Frank [0000-0001-6399-9497], Qureshi, Nusrat [0000-0002-5753-5984], Richter, Christian [0000-0002-5420-2826], Schlundt, Andreas [0000-0003-2254-7560], Weigand, Julia E. [0000-0003-4247-1348], Wirmer-Bartoschek, Julia [0000-0002-0642-1311], Schwalbe, Harald [0000-0001-5693-7909], Berg, Hannes, Wirtz Martin, Maria A., Altincekic, Nadide, Alshamleh, Islam, Kaur Bains, Jasleen, Blechar, Julius, Ceylan, Betül, Jesus, Vanessa de, Dhamotharan, Karthikeyan, Fuks, Christin, Gande, Santosh L., Hargittay, Bruno, Hohmann, Katharina F., Hutchison, Marie T., Marianne Korn, Sophie, Krishnathas, Robin, Kutz, Felicitas, Linhard, Verena, Matzel, Tobias, Meiser, Nathalie, Niesteruk, Anna, Pyper, Dennis J., Schulte, Linda, Trucks, Sven, Azzaoui, Kamal, Blommers, Marcel J.J., Gadiya, Yojana, Karki, Reagon, Zaliani, Andrea, Gribbon, Philip, da Silva Almeida, Marcius, Dinis Anobom, Cristiane, Bula, Anna L., Bütikofer, Matthias, Putinhon Caruso, Ícaro, Caterina Felli, Isabella, Da Poian, Andrea T., Cardoso de Amorim, Gisele, Fourkiotis, Nikolaos K., Gallo, Angelo, Ghosh, Dhiman, Gomes-Neto, Francisco, Gorbatyuk, Oksana, Hao, Bing, Kurauskas, Vilius, Lecoq, Lauriane, Li, Yunfeng, Cunha Mebus-Antunes, Nathane, Mompeán, Miguel, Cristtina Neves-Martins, Thais, Ninot-Pedrosa, Martí, Pinheiro, Anderson S.., Pontoriero, Letizia, Pustovalova, Yulia, Riek, Roland, Robertson, Angus J., Jose Abi Saad, Marie, Treviño, Miguel A., Tsika, Aikaterini C., Almeida, Fabio C.L., Bax, Ad, Henzler-Wildman, Katherine, Hoch, Jeffrey C., Jaudzems, Kristaps, Laurents, Douglas V., Orts, Julien, Pierattelli, Roberta, Spyroulias, Georgios A., Duchardt-Ferner, Elke, Ferner, Jan, Fürtig, Boris, Hengesbach, Martin, Löhr, Frank, Qureshi, Nusrat, Richter, Christian, Saxena, Krishna, Schlundt, Andreas, Sreeramulu, Sridhar, Wacker, Anna, Weigand, Julia E., Wirmer-Bartoschek, Julia, Wöhnert, Jens, and Schwalbe, Harald

Abstract

SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.

Published
2022

27. Experimental Characterization of the Hepatitis B Virus Capsid Dynamics by Solid-State NMR

Author

Malär, Alexander A., Callon, Morgane, Smith, Albert A., Wang, Shishan, Lecoq, Lauriane, Pérez-Segura, Carolina, Hadden-Perilla, Jodi A., Böckmann, Anja, and Meier, Beat H.

Subjects
relaxation, solid-state NMR, virus, dynamics, molecular dynamics, QH301-705.5, Molecular Biosciences, Biology (General), Original Research
Abstract

Protein plasticity and dynamics are important aspects of their function. Here we use solid-state NMR to experimentally characterize the dynamics of the 3.5 MDa hepatitis B virus (HBV) capsid, assembled from 240 copies of the Cp149 core protein. We measure both T1 and T1ρ relaxation times, which we use to establish detectors on the nanosecond and microsecond timescale. We compare our results to those from a 1 microsecond all-atom Molecular Dynamics (MD) simulation trajectory for the capsid. We show that, for the constituent residues, nanosecond dynamics are faithfully captured by the MD simulation. The calculated values can be used in good approximation for the NMR-non-detected residues, as well as to extrapolate into the range between the nanosecond and microsecond dynamics, where NMR has a blind spot at the current state of technology. Slower motions on the microsecond timescale are difficult to characterize by all-atom MD simulations owing to computational expense, but are readily accessed by NMR. The two methods are, thus, complementary, and a combination thereof can reliably characterize motions covering correlation times up to a few microseconds., Frontiers in Molecular Biosciences, 8, ISSN:2296-889X

Published
2022
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30. Pharmacomodulation of a ligand targeting the HBV capsid hydrophobic pocket

Author

Briday, Mathilde, primary, Hallé, François, additional, Lecoq, Lauriane, additional, Radix, Sylvie, additional, Martin, Juliette, additional, Montserret, Roland, additional, Dujardin, Marie, additional, Fogeron, Marie-Laure, additional, Nassal, Michael, additional, Meier, Beat H., additional, Lomberget, Thierry, additional, and Böckmann, Anja, additional

Published
2022
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32. Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

Author

Altincekic, Nadide, Korn, Sophie Marianne, Qureshi, Nusrat Shahin, Dujardin, Marie, Ninot-Pedrosa, Martí, Abele, Rupert, Abi Saad, Marie Jose, Alfano, Caterina, Almeida, Fabio, Alshamleh, Islam, de Amorim, Gisele Cardoso, Anderson, Thomas, Anobom, Cristiane, Anorma, Chelsea, Bains, Jasleen Kaur, Bax, Adriaan, Blackledge, Martin, Blechar, Julius, Böckmann, Anja, Brigandat, Louis, Bula, Anna, Bütikofer, Matthias, Camacho-Zarco, Aldo, Carlomagno, Teresa, Caruso, Icaro Putinhon, Ceylan, Betül, Chaikuad, Apirat, Chu, Feixia, Cole, Laura, Crosby, Marquise, de Jesus, Vanessa, Dhamotharan, Karthikeyan, Felli, Isabella, Ferner, Jan, Fleischmann, Yanick, Fogeron, Marie-Laure, Fourkiotis, Nikolaos, Fuks, Christin, Fürtig, Boris, Gallo, Angelo, Gande, Santosh, Gerez, Juan Atilio, Ghosh, Dhiman, GOMES-NETO, Francisco, Gorbatyuk, Oksana, Guseva, Serafima, Hacker, Carolin, Häfner, Sabine, Hao, Bing, Hargittay, Bruno, Henzler-Wildman, K., Hoch, Jeffrey, Hohmann, Katharina, Hutchison, Marie, Jaudzems, Kristaps, Jović, Katarina, Kaderli, Janina, Kalniņš, Gints, Kaņepe, Iveta, Kirchdoerfer, Robert, Kirkpatrick, John, Knapp, Stefan, Krishnathas, Robin, Kutz, Felicitas, zur Lage, Susanne, Lambertz, Roderick, Lang, Andras, Laurents, Douglas, Lecoq, Lauriane, Linhard, Verena, Löhr, Frank, Malki, Anas, Bessa, Luiza Mamigonian, Martin, Rachel, Matzel, Tobias, Maurin, Damien, McNutt, Seth, Mebus-Antunes, Nathane Cunha, Meier, Beat, Meiser, Nathalie, Mompeán, Miguel, Monaca, Elisa, Montserret, Roland, Mariño Perez, Laura, Moser, Celine, Muhle-Goll, Claudia, Neves-Martins, Thais Cristtina, Ni, Xiamonin, Norton-Baker, Brenna, Pierattelli, Roberta, Pontoriero, Letizia, Pustovalova, Yulia, Ohlenschläger, Oliver, Orts, Julien, Da Poian, Andrea, Pyper, Dennis, Richter, Christian, Riek, Roland, Rienstra, Chad, Robertson, Angus, Pinheiro, Anderson, Sabbatella, Raffaele, Salvi, Nicola, Saxena, Krishna, Schulte, Linda, Schiavina, Marco, Schwalbe, Harald, Silber, Mara, Almeida, Marcius da Silva, Sprague-Piercy, Marc, Spyroulias, Georgios, Sreeramulu, Sridhar, Tants, Jan-Niklas, Tārs, Kaspars, Torres, Felix, Töws, Sabrina, Treviño, Miguel, Trucks, Sven, Tsika, Aikaterini, Varga, Krisztina, Wang, Ying, Weber, Marco, Weigand, Julia, Wiedemann, Christoph, Wirmer-Bartoschek, Julia, Wirtz Martin, Maria Alexandra, Zehnder, Johannes, Hengesbach, Martin, Schlundt, Andreas, Treviño, Miguel Á., Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance (BMRZ), Microbiologie moléculaire et biochimie structurale / Molecular Microbiology and Structural Biochemistry (MMSB), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), Goethe University Frankfurt am Main, German Research Foundation, Cassa di Risparmio di Firenze, European Commission, University of New Hampshire, The Free State of Thuringia, National Institutes of Health (US), National Science Foundation (US), Howard Hughes Medical Institute, Latvian Council of Science, Ministry of Development and Investments (Greece), Helmholtz Association, Centre National de la Recherche Scientifique (France), Agence Nationale de la Recherche (France), Fondation pour la Recherche Médicale, Swiss National Science Foundation, Fonds National Suisse de la Recherche Scientifique, ETH Zurich, European Research Council, Université Grenoble Alpes, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Fundación 'la Caixa', Instituto de Salud Carlos III, Boehringer Ingelheim Fonds, Ministero dell'Istruzione, dell'Università e della Ricerca, Polytechnic Foundation of Frankfurt am Main, Goethe University Frankfurt, CNRS/Lyon University, Fondazione Ri.MED, Federal University of Rio de Janeiro, Caxias Federal University of Rio de Janeiro, University of Wisconsin-Madison, University of California, NIDDK, IBS, Latvian Institute of Organic Synthesis, Leibniz University Hannover, Helmholtz Centre for Infection Research, Universidade Estadual Paulista (Unesp), Buchmann Institute for Molecular Life Sciences, University of Florence, University of Patras, Oswaldo Cruz Foundation (FIOCRUZ), UConn Health, Signals GmbH Co. KG, Leibniz Institute on Aging—Fritz Lipmann Institute (FLI), Latvian Biomedical Research and Study Centre, Spanish National Research Council (CSIC), Karlsruhe Institute of Technology, Technical University of Darmstadt, Martin Luther University Halle-Wittenberg, Altincekic N., Korn S.M., Qureshi N.S., Dujardin M., Ninot-Pedrosa M., Abele R., Abi Saad M.J., Alfano C., Almeida F.C.L., Alshamleh I., de Amorim G.C., Anderson T.K., Anobom C.D., Anorma C., Bains J.K., Bax A., Blackledge M., Blechar J., Bockmann A., Brigandat L., Bula A., Butikofer M., Camacho-Zarco A.R., Carlomagno T., Caruso I.P., Ceylan B., Chaikuad A., Chu F., Cole L., Crosby M.G., de Jesus V., Dhamotharan K., Felli I.C., Ferner J., Fleischmann Y., Fogeron M.-L., Fourkiotis N.K., Fuks C., Furtig B., Gallo A., Gande S.L., Gerez J.A., Ghosh D., Gomes-Neto F., Gorbatyuk O., Guseva S., Hacker C., Hafner S., Hao B., Hargittay B., Henzler-Wildman K., Hoch J.C., Hohmann K.F., Hutchison M.T., Jaudzems K., Jovic K., Kaderli J., Kalnins G., Kanepe I., Kirchdoerfer R.N., Kirkpatrick J., Knapp S., Krishnathas R., Kutz F., zur Lage S., Lambertz R., Lang A., Laurents D., Lecoq L., Linhard V., Lohr F., Malki A., Bessa L.M., Martin R.W., Matzel T., Maurin D., McNutt S.W., Mebus-Antunes N.C., Meier B.H., Meiser N., Mompean M., Monaca E., Montserret R., Marino Perez L., Moser C., Muhle-Goll C., Neves-Martins T.C., Ni X., Norton-Baker B., Pierattelli R., Pontoriero L., Pustovalova Y., Ohlenschlager O., Orts J., Da Poian A.T., Pyper D.J., Richter C., Riek R., Rienstra C.M., Robertson A., Pinheiro A.S., Sabbatella R., Salvi N., Saxena K., Schulte L., Schiavina M., Schwalbe H., Silber M., Almeida M.D.S., Sprague-Piercy M.A., Spyroulias G.A., Sreeramulu S., Tants J.-N., Tars K., Torres F., Tows S., Trevino M.A., Trucks S., Tsika A.C., Varga K., Wang Y., Weber M.E., Weigand J.E., Wiedemann C., Wirmer-Bartoschek J., Wirtz Martin M.A., Zehnder J., Hengesbach M., Schlundt A., HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany., and Obra Social la Caixa

Subjects
Life sciences, biology, SARS-COV-2, COVID-19, protein production, structural biology, NMR, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Biochemistry, Genetics and Molecular Biology (miscellaneous), Biochemistry, Accessory proteins, NMR spectroscopy, ddc:570, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Molecular Biosciences, ddc:610, Nonstructural proteins, Molecular Biology, Original Research, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], SARS-CoV-2, Intrinsically disordered region, nonstructural proteins, structural proteins, Cell-free protein synthesis, intrinsically disordered region, cell-free protein synthesis, accessory proteins, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Structural proteins
Abstract

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form., This work was supported by Goethe University (Corona funds), the DFG-funded CRC: “Molecular Principles of RNA-Based Regulation,” DFG infrastructure funds (project numbers: 277478796, 277479031, 392682309, 452632086, 70653611), the state of Hesse (BMRZ), the Fondazione CR Firenze (CERM), and the IWB-EFRE-program 20007375. This project has received funding from the European Union’s Horizon 2020 research and innovation program under Grant Agreement No. 871037. AS is supported by DFG Grant SCHL 2062/2-1 and by the JQYA at Goethe through project number 2019/AS01. Work in the lab of KV was supported by a CoRE grant from the University of New Hampshire. The FLI is a member of the Leibniz Association (WGL) and financially supported by the Federal Government of Germany and the State of Thuringia. Work in the lab of RM was supported by NIH (2R01EY021514) and NSF (DMR-2002837). BN-B was supported by theNSF GRFP.MCwas supported byNIH (R25 GM055246 MBRS IMSD), and MS-P was supported by the HHMI Gilliam Fellowship. Work in the labs of KJ and KT was supported by Latvian Council of Science Grant No. VPP-COVID 2020/1-0014. Work in the UPAT’s lab was supported by the INSPIRED (MIS 5002550) project, which is implemented under the Action “Reinforcement of the Research and Innovation Infrastructure,” funded by the Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and cofinanced by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011- 285950–“SEE-DRUG” project (purchase of UPAT’s 700MHz NMR equipment). Work in the CM-G lab was supported by the Helmholtz society. Work in the lab of ABö was supported by the CNRS, the French National Research Agency (ANR, NMRSCoV2- ORF8), the Fondation de la Recherche Médicale (FRM, NMR-SCoV2-ORF8), and the IR-RMN-THC Fr3050 CNRS. Work in the lab of BM was supported by the Swiss National Science Foundation (Grant number 200020_188711), the Günthard Stiftung für Physikalische Chemie, and the ETH Zurich. Work in the labs of ABö and BM was supported by a common grant from SNF (grant 31CA30_196256). This work was supported by the ETHZurich, the grant ETH40 18 1, and the grant Krebsliga KFS 4903 08 2019. Work in the lab of the IBS Grenoble was supported by the Agence Nationale de Recherche (France) RA-COVID SARS2NUCLEOPROTEIN and European Research Council Advanced Grant DynamicAssemblies. Work in the CA lab was supported by Patto per il Sud della Regione Siciliana–CheMISt grant (CUP G77B17000110001). Part of this work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-05-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE- 0003). Work at the UW-Madison was supported by grant numbers NSF MCB2031269 and NIH/NIAID AI123498. MM is a Ramón y Cajal Fellow of the Spanish AEI-Ministry of Science and Innovation (RYC2019-026574-I), and a “La Caixa” Foundation (ID 100010434) Junior Leader Fellow (LCR/BQ/PR19/11700003). Funded by project COV20/00764 fromthe Carlos III Institute of Health and the SpanishMinistry of Science and Innovation to MMand DVL. VDJ was supported by the Boehringer Ingelheim Fonds. Part of this work used the resources of the Italian Center of Instruct-ERIC at the CERM/ CIRMMP infrastructure, supported by the Italian Ministry for University and Research (FOE funding). CF was supported by the Stiftung Polytechnische Gesellschaft. Work in the lab of JH was supported by NSF (RAPID 2030601) and NIH (R01GM123249).

Published
2021
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33. Temperature-Dependent Solid-State NMR Proton Chemical-Shift Values and Hydrogen Bonding

Author

Malär, Alexander A., Völker, Laura A., Cadalbert, Riccardo, Lecoq, Lauriane, Ernst, Matthias, Böckmann, Anja, Meier, Beat H., and Wiegand, Thomas

Subjects
Quantitative Biology::Biomolecules, Magnetic Resonance Spectroscopy, Cryoelectron Microscopy, Temperature, Hydrogen Bonding, Protons, Article, 3. Good health
Abstract

Temperature-dependent NMR experiments are often complicated by rather long magnetic-field equilibration times, for example, occurring upon a change of sample temperature. We demonstrate that the fast temporal stabilization of a magnetic field can be achieved by actively stabilizing the temperature of the magnet bore, which allows quantification of the weak temperature dependence of a proton chemical shift, which can be diagnostic for the presence of hydrogen bonds. Hydrogen bonding plays a central role in molecular recognition events from both fields, chemistry and biology. Their direct detection by standard structure-determination techniques, such as X-ray crystallography or cryo-electron microscopy, remains challenging due to the difficulties of approaching the required resolution, on the order of 1 Å. We, herein, explore a spectroscopic approach using solid-state NMR to identify protons engaged in hydrogen bonds and explore the measurement of proton chemical-shift temperature coefficients. Using the examples of a phosphorylated amino acid and the protein ubiquitin, we show that fast magic-angle spinning (MAS) experiments at 100 kHz yield sufficient resolution in proton-detected spectra to quantify the rather small chemical-shift changes upon temperature variations. ISSN:1520-6106 ISSN:1520-5207 ISSN:1089-5647

Published
2021
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35. 1H, 15N and 13C backbone and side chain solution NMR assignments of the truncated small hepatitis delta antigen Δ60-S-HDAg.

Author

Yang, Yang, Delcourte, Loïc, Fogeron, Marie-Laure, Böckmann, Anja, and Lecoq, Lauriane

Abstract

Hepatitis D virus (HDV) is a defective virus that relies on hepatitis B virus envelope proteins to complete its replication cycle. The HDV genome contains two isoforms of hepatitis delta antigen: the small and the large hepatitis delta antigens (S- and L-HDAg). Here we report the 1H, 13C and 15 N backbone and side chain resonance assignments of an N-terminally truncated form of S-HDAg (SΔ60), which lacks the 1–60 oligomerization domain. We derived secondary structures based on NMR chemical shifts, which will be used in further structural and functional studies. We show that SΔ60 is partially disordered, and that the central structured part contains two well-defined α-helices of 22 and 17 residues, respectively. A temperature titration allowed to identify the residues involved in hydrogen bonds. [ABSTRACT FROM AUTHOR]

Published
2022
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36. Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

Author

Altınçekiç, Nadide, Korn, Sophie Marianne, Qureshi, Nusrat Shahin, Dujardin, Marie, Ninot-Pedrosa, Martí, Abele, Rupert, Abi Saad, Marie Jose, Alfano, Caterina, Almeida, Fabio C. L., Alshamleh, Islam, Amorim, Gisele Cardoso de, Anderson, Thomas K., Anobom, Cristiane D., Anorma, Chelsea, Bains, Jasleen Kaur, Bax, Adriaan, Blackledge, Martin, Blechar, Julius, Böckmann, Anja, Brigandat, Louis, Bula, Anna, Bütikofer, Matthias, Camacho-Zarco, Aldo R., Carlomagno, Teresa, Caruso, Icaro Putinhon, Ceylan, Betül, Chaikuad, Apirat, Chu, Feixia, Cole, Laura, Crosby, Marquise G., De Jesus, Vanessa, Dhamotharan, Karthikeyan, Felli, Isabella C., Ferner, Jan, Fleischmann, Yanick, Fogeron, Marie-Laure, Fourkiotis, Nikolaos K., Fuks, Christin, Fürtig, Boris, Gallo, Angelo, Gande, Santosh L., Gerez, Juan Atilio, Ghosh, Dhiman, Gomes-Neto, Francisco, Gorbatyuk, Oksana, Guseva, Serafima, Hacker, Carolin, Häfner, Sabine, Hao, Bing, Hargittay, Bruno, Henzler-Wildman, Katherine, Hoch, Jeffrey C., Hohmann, Katharina Felicitas, Hutchison, Marie T., Jaudzems, Kristaps, Jović, Katarina, Kaderli, Janina, Kalnins, Gints, Kanepe, Iveta, Kirchdoerfer, Robert N., Kirkpatrick, John, Knapp, Stefan, Krishnathas, Robin, Kutz, Felicitas, Zur Lage, Susanne, Lambertz, Roderick, Lang, Andras, Laurents, Douglas, Lecoq, Lauriane, Linhard, Verena, Löhr, Frank, Malki, Anas, Bessa, Luiza Mamigonian, Martin, Rachel W., Matzel, Tobias, Maurin, Damien, McNutt, Seth W., Mebus-Antunes, Nathane Cunha, Meier, Beat H., Meiser, Nathalie, Mompeán, Miguel, Monaca, Elisa, Montserret, Roland, Perez, Laura Mariño, Moser, Celine, Muhle-Goll, Claudia, Neves-Martins, Thais Cristtina, Ni, Xiamonin, Norton-Baker, Brenna, Pierattelli, Roberta, Pontoriero, Letizia, Pustovalova, Yulia, Ohlenschläger, Oliver, Orts, Julien, Poian, Andrea T. da, Pyper, Dennis Joshua, Richter, Christian, Riek, Roland, Rienstra, Chad M., Robertson, Angus, Pinheiro, Anderson S., Sabbatella, Raffaele, Salvi, Nicola, Saxena, Krishna, Schulte, Linda, Schiavina, Marco, Schwalbe, Harald, Silber, Mara, Almeida, Marcius da Silva, Sprague-Piercy, Marc A., Spyroulias, Georgios A., Sreeramulu, Sridhar, Tants, Jan-Niklas, Tars, Kaspars, Torres, Felix, Töws, Sabrina, Trevino, Miguel A., Trucks, Sven, Tsika, Aikaterini C., Varga, Krisztina, Wang, Ying, Weber, Marco E., Weigand, Julia E., Wiedemann, Christoph, Wirmer-Bartoschek, Julia, Wirtz Martin, Maria Alexandra, Zehnder, Johannes, Hengesbach, Martin, Schlundt, Andreas, Altınçekiç, Nadide, Korn, Sophie Marianne, Qureshi, Nusrat Shahin, Dujardin, Marie, Ninot-Pedrosa, Martí, Abele, Rupert, Abi Saad, Marie Jose, Alfano, Caterina, Almeida, Fabio C. L., Alshamleh, Islam, Amorim, Gisele Cardoso de, Anderson, Thomas K., Anobom, Cristiane D., Anorma, Chelsea, Bains, Jasleen Kaur, Bax, Adriaan, Blackledge, Martin, Blechar, Julius, Böckmann, Anja, Brigandat, Louis, Bula, Anna, Bütikofer, Matthias, Camacho-Zarco, Aldo R., Carlomagno, Teresa, Caruso, Icaro Putinhon, Ceylan, Betül, Chaikuad, Apirat, Chu, Feixia, Cole, Laura, Crosby, Marquise G., De Jesus, Vanessa, Dhamotharan, Karthikeyan, Felli, Isabella C., Ferner, Jan, Fleischmann, Yanick, Fogeron, Marie-Laure, Fourkiotis, Nikolaos K., Fuks, Christin, Fürtig, Boris, Gallo, Angelo, Gande, Santosh L., Gerez, Juan Atilio, Ghosh, Dhiman, Gomes-Neto, Francisco, Gorbatyuk, Oksana, Guseva, Serafima, Hacker, Carolin, Häfner, Sabine, Hao, Bing, Hargittay, Bruno, Henzler-Wildman, Katherine, Hoch, Jeffrey C., Hohmann, Katharina Felicitas, Hutchison, Marie T., Jaudzems, Kristaps, Jović, Katarina, Kaderli, Janina, Kalnins, Gints, Kanepe, Iveta, Kirchdoerfer, Robert N., Kirkpatrick, John, Knapp, Stefan, Krishnathas, Robin, Kutz, Felicitas, Zur Lage, Susanne, Lambertz, Roderick, Lang, Andras, Laurents, Douglas, Lecoq, Lauriane, Linhard, Verena, Löhr, Frank, Malki, Anas, Bessa, Luiza Mamigonian, Martin, Rachel W., Matzel, Tobias, Maurin, Damien, McNutt, Seth W., Mebus-Antunes, Nathane Cunha, Meier, Beat H., Meiser, Nathalie, Mompeán, Miguel, Monaca, Elisa, Montserret, Roland, Perez, Laura Mariño, Moser, Celine, Muhle-Goll, Claudia, Neves-Martins, Thais Cristtina, Ni, Xiamonin, Norton-Baker, Brenna, Pierattelli, Roberta, Pontoriero, Letizia, Pustovalova, Yulia, Ohlenschläger, Oliver, Orts, Julien, Poian, Andrea T. da, Pyper, Dennis Joshua, Richter, Christian, Riek, Roland, Rienstra, Chad M., Robertson, Angus, Pinheiro, Anderson S., Sabbatella, Raffaele, Salvi, Nicola, Saxena, Krishna, Schulte, Linda, Schiavina, Marco, Schwalbe, Harald, Silber, Mara, Almeida, Marcius da Silva, Sprague-Piercy, Marc A., Spyroulias, Georgios A., Sreeramulu, Sridhar, Tants, Jan-Niklas, Tars, Kaspars, Torres, Felix, Töws, Sabrina, Trevino, Miguel A., Trucks, Sven, Tsika, Aikaterini C., Varga, Krisztina, Wang, Ying, Weber, Marco E., Weigand, Julia E., Wiedemann, Christoph, Wirmer-Bartoschek, Julia, Wirtz Martin, Maria Alexandra, Zehnder, Johannes, Hengesbach, Martin, and Schlundt, Andreas

Abstract

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.

Published
2021

37. Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

Author

Goethe University Frankfurt am Main, German Research Foundation, Cassa di Risparmio di Firenze, European Commission, University of New Hampshire, The Free State of Thuringia, National Institutes of Health (US), National Science Foundation (US), Howard Hughes Medical Institute, Latvian Council of Science, Ministry of Development and Investments (Greece), Helmholtz Association, Centre National de la Recherche Scientifique (France), Agence Nationale de la Recherche (France), Fondation pour la Recherche Médicale, Swiss National Science Foundation, Fonds National Suisse de la Recherche Scientifique, ETH Zurich, European Research Council, Université Grenoble Alpes, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Fundación la Caixa, Instituto de Salud Carlos III, Boehringer Ingelheim Fonds, Ministero dell'Istruzione, dell'Università e della Ricerca, Polytechnic Foundation of Frankfurt am Main, Altincekic, Nadide, Korn, Sophie Marianne, Qureshi, Nusrat Shahin, Dujardin, Marie, Ninot-Pedrosa, Martí, Abele, Rupert, Abi Saad, Marie Jose, Alfano, Caterina, Almeida, Fabio C. L., Alshamleh, Islam, Cardoso de Amorim, Gisele, Anorma, Chelsea, Bains, Jasleen Kaur, Bax, Andriaan, Blackledge, Martin, Blechar, Julius, Böckmann, Anja, Brigandat, Louis, Bula, Anna, Bütikofer, Matthias, Camacho-Zarco, Aldo R., Ghosh, Dhiman, Carlomagno, Teresa, Caruso, Icaro Putinhon, Ceylan, Betül, Chaikuad, Apirat, Chu, Feixia, Cole, Laura, Crosby, Marquise G., Jesus, Vanessa de, Dhamotharan, Karthikeyan, Felli, Isabella C., Gomes-Neto, Francisco, Ferner, Jan, Fleischmann, Yanick, Fogeron, Marie-Laure, Fourkiotis, Nikolaos K., Fuks, Christin, Fürtig, Boris, Gallo, Angelo, Gande, Santosh L., Gerez, Juan Atilio, Gorbatyuk, Oksana, Guseva, Serafima, Hacker, Carolin, Häfner, Sabine, Hao, Bing, Hargittay, Bruno, Henzler-Wildman, K., Hoch, Jeffrey C., Malki, Anas, Hohmann, Katharina F., Hutchison, Marie T., Jaudzems, Kristaps, Jović, Katarina, Kaderli, Janina, Kalniņš, Gints, Kaņepe, Iveta, Kirchdoerfer, Robert N., Kirkpatrick, John, Knapp, Stefan, Bessa, Luiza Mamigonian, Krishnathas, Robin, Kutz, Felicitas, Lage, Susanne zur, Lambertz, Roderick, Lang, Andras, Laurents, Douglas V., Lecoq, Lauriane, Linhard, Verena, Löhr, Frank, Martin, Rachel W., Matzel, Tobias, Maurin, Damien, McNutt, Seth W., Mebus-Antunes, Nathane Cunha, Meier, Beat H., Meiser, Nathalie, Mompeán, Miguel, Pinheiro, Anderson S.., Monaca, Elisa, Montserret, Roland, Mariño Perez, Laura, Moser, Celine, Muhle-Goll, Claudia, Neves-Martins, Thais Cristtina, Ni, Xiamonin, Norton-Baker, Brenna, Pierattelli, Roberta, Pontoriero, Letizia, Sabbatella, Raffaele, Pustovalova, Yulia, Ohlenschläger, Oliver, Orts, Julien, Da Poian, Andrea T., Pyper, Dennis J., Richter, Christian, Riek, Roland, Rienstra, Chad M., Robertson, Angus, Salvi, Nicola, Saxena, Krishna, Schulte, Linda, Schiavina, Marco, Schwalbe, Harald, Silber, Mara, Silva Almeida, Marcius da, Sprague-Piercy, Marc A., Anderson, Thomas K., Spyroulias, Georgios A., Sreeramulu, Sridhar, Tants, Jan-Niklas, Tārs, Kaspars, Torres, Felix, Töws, Sabrina, Treviño, Miguel A., Trucks, Sven, Tsika, Aikaterini C., Varga, Krisztina, Anobom, Cristiane D., Wang, Ying, Weber, Marco E., Weigand, Julia E., Wiedemann, Christoph, Wirmer-Bartoschek, Julia, Wirtz Martin, Maria Alexandra, Zehnder, Johannes, Hengesbach, Martin, Schlundt, Andreas, Goethe University Frankfurt am Main, German Research Foundation, Cassa di Risparmio di Firenze, European Commission, University of New Hampshire, The Free State of Thuringia, National Institutes of Health (US), National Science Foundation (US), Howard Hughes Medical Institute, Latvian Council of Science, Ministry of Development and Investments (Greece), Helmholtz Association, Centre National de la Recherche Scientifique (France), Agence Nationale de la Recherche (France), Fondation pour la Recherche Médicale, Swiss National Science Foundation, Fonds National Suisse de la Recherche Scientifique, ETH Zurich, European Research Council, Université Grenoble Alpes, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Fundación la Caixa, Instituto de Salud Carlos III, Boehringer Ingelheim Fonds, Ministero dell'Istruzione, dell'Università e della Ricerca, Polytechnic Foundation of Frankfurt am Main, Altincekic, Nadide, Korn, Sophie Marianne, Qureshi, Nusrat Shahin, Dujardin, Marie, Ninot-Pedrosa, Martí, Abele, Rupert, Abi Saad, Marie Jose, Alfano, Caterina, Almeida, Fabio C. L., Alshamleh, Islam, Cardoso de Amorim, Gisele, Anorma, Chelsea, Bains, Jasleen Kaur, Bax, Andriaan, Blackledge, Martin, Blechar, Julius, Böckmann, Anja, Brigandat, Louis, Bula, Anna, Bütikofer, Matthias, Camacho-Zarco, Aldo R., Ghosh, Dhiman, Carlomagno, Teresa, Caruso, Icaro Putinhon, Ceylan, Betül, Chaikuad, Apirat, Chu, Feixia, Cole, Laura, Crosby, Marquise G., Jesus, Vanessa de, Dhamotharan, Karthikeyan, Felli, Isabella C., Gomes-Neto, Francisco, Ferner, Jan, Fleischmann, Yanick, Fogeron, Marie-Laure, Fourkiotis, Nikolaos K., Fuks, Christin, Fürtig, Boris, Gallo, Angelo, Gande, Santosh L., Gerez, Juan Atilio, Gorbatyuk, Oksana, Guseva, Serafima, Hacker, Carolin, Häfner, Sabine, Hao, Bing, Hargittay, Bruno, Henzler-Wildman, K., Hoch, Jeffrey C., Malki, Anas, Hohmann, Katharina F., Hutchison, Marie T., Jaudzems, Kristaps, Jović, Katarina, Kaderli, Janina, Kalniņš, Gints, Kaņepe, Iveta, Kirchdoerfer, Robert N., Kirkpatrick, John, Knapp, Stefan, Bessa, Luiza Mamigonian, Krishnathas, Robin, Kutz, Felicitas, Lage, Susanne zur, Lambertz, Roderick, Lang, Andras, Laurents, Douglas V., Lecoq, Lauriane, Linhard, Verena, Löhr, Frank, Martin, Rachel W., Matzel, Tobias, Maurin, Damien, McNutt, Seth W., Mebus-Antunes, Nathane Cunha, Meier, Beat H., Meiser, Nathalie, Mompeán, Miguel, Pinheiro, Anderson S.., Monaca, Elisa, Montserret, Roland, Mariño Perez, Laura, Moser, Celine, Muhle-Goll, Claudia, Neves-Martins, Thais Cristtina, Ni, Xiamonin, Norton-Baker, Brenna, Pierattelli, Roberta, Pontoriero, Letizia, Sabbatella, Raffaele, Pustovalova, Yulia, Ohlenschläger, Oliver, Orts, Julien, Da Poian, Andrea T., Pyper, Dennis J., Richter, Christian, Riek, Roland, Rienstra, Chad M., Robertson, Angus, Salvi, Nicola, Saxena, Krishna, Schulte, Linda, Schiavina, Marco, Schwalbe, Harald, Silber, Mara, Silva Almeida, Marcius da, Sprague-Piercy, Marc A., Anderson, Thomas K., Spyroulias, Georgios A., Sreeramulu, Sridhar, Tants, Jan-Niklas, Tārs, Kaspars, Torres, Felix, Töws, Sabrina, Treviño, Miguel A., Trucks, Sven, Tsika, Aikaterini C., Varga, Krisztina, Anobom, Cristiane D., Wang, Ying, Weber, Marco E., Weigand, Julia E., Wiedemann, Christoph, Wirmer-Bartoschek, Julia, Wirtz Martin, Maria Alexandra, Zehnder, Johannes, Hengesbach, Martin, and Schlundt, Andreas

Abstract

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.

Published
2021

38. Biomolecular solid-state NMR spectroscopy at highest field: the gain in resolution at 1200 MHz

Author

Callon, Morgane, primary, Malär, Alexander A., additional, Pfister, Sara, additional, Rímal, Václav, additional, Weber, Marco E., additional, Wiegand, Thomas, additional, Zehnder, Johannes, additional, Chávez, Matías, additional, Deb, Rajdeep, additional, Cadalbert, Riccardo, additional, Däpp, Alexander, additional, Fogeron, Marie-Laure, additional, Hunkeler, Andreas, additional, Lecoq, Lauriane, additional, Torosyan, Anahit, additional, Zyla, Dawid, additional, Glockshuber, Rudolf, additional, Jonas, Stefanie, additional, Nassal, Michael, additional, Ernst, Matthias, additional, Böckmann, Anja, additional, and Meier, Beat H., additional

Published
2021
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43. Protein NMR Spectroscopy at 150 kHz Magic‐Angle Spinning Continues To Improve Resolution and Mass Sensitivity

Author

Schledorn, Maarten, primary, Malär, Alexander A., additional, Torosyan, Anahit, additional, Penzel, Susanne, additional, Klose, Daniel, additional, Oss, Andres, additional, Org, Mai‐Liis, additional, Wang, Shishan, additional, Lecoq, Lauriane, additional, Cadalbert, Riccardo, additional, Samoson, Ago, additional, Böckmann, Anja, additional, and Meier, Beat H., additional

Published
2020
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44. 100 kHz MAS Proton-Detected NMR Spectroscopy of Hepatitis B Virus Capsids

Author

Lecoq, Lauriane, Schledorn, Maarten, Wang, Shishan, Smith-Penzel, Susanne, Malär, Alexander, Callon, Morgane, Nassal, Michael, Meier, Beat, Böckmann, Anja, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Microbiologie moléculaire et biochimie structurale / Molecular Microbiology and Structural Biochemistry (MMSB), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), University Hospital Freiburg, Physical Chemistry [ETH Zürich], Department of Chemistry and Applied Biosciences [ETH Zürich] (D-CHAB), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich)- Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
deuteration, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Brief Research Report, fast MAS, Solid-state NMR, Fast MAS, Proton detection, Carbon detection, Deuteration, Hepatitis B virus, Capsid, Core protein, proton detection, lcsh:Biology (General), [CHIM.ANAL]Chemical Sciences/Analytical chemistry, carbon detection, core protein, capsid, solid-state NMR, Molecular Biosciences, hepatitis B virus, lcsh:QH301-705.5
Abstract

International audience; We sequentially assigned the fully-protonated capsids made from core proteins of the Hepatitis B virus using proton detection at 100 kHz magic-angle spinning (MAS) in 0.7 mm rotors and compare sensitivity and assignment completeness to previously obtained assignments using carbon-detection techniques in 3.2 mm rotors and 17.5 kHz MAS. We show that proton detection shows a global gain of a factor ∼50 in mass sensitivity, but that signal-to-noise ratios and completeness of the assignment was somewhat higher for carbon-detected experiments for comparable experimental times. We also show that deuteration and H N back protonation improves the proton linewidth at 100 kHz MAS by a factor of 1.5, from an average of 170-110 Hz, and by a factor of 1.3 compared to deuterated capsids at 60 kHz MAS in a 1.3 mm rotor. Yet, several H N protons cannot be back-exchanged due to solvent inaccessibility, which results in a total of 15% of the amides missing in the spectra.

Published
2019
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45. Combining Cell-Free Protein Synthesis and NMR Into a Tool to Study Capsid Assembly Modulation

Author

Wang, Shishan, Fogeron, Marie-Laure, Schledorn, Maarten, Dujardin, Marie, Penzel, Susanne, Burdette, Dara, Berke, Jan, Nassal, Michael, Lecoq, Lauriane, Meier, Beat, Böckmann, Anja, Microbiologie moléculaire et biochimie structurale / Molecular Microbiology and Structural Biochemistry (MMSB), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Physical Chemistry [Zurich, Switzerland], Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), Gilead Sciences, Inc. [Foster City, CA, USA], Janssen Pharmaceutica N.V. [Beerse, Belgium], Internal Medicine II/Molecular Biology [Freiburg, Germany], University Hospital Freiburg, and Bockmann, Anja

Subjects
[CHIM.ANAL] Chemical Sciences/Analytical chemistry, HBV-hepatitis B virus, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], viruses, cell-free protein synthesis, NMR, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], proton detection, capsid, assembly modulation, lcsh:Biology (General), [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Molecular Biosciences, lcsh:QH301-705.5, HBV—hepatitis B virus, Original Research
Abstract

International audience; Modulation of capsid assembly by small molecules has become a central concept in the fight against viral infection. Proper capsid assembly is crucial to form the high molecular weight structures that protect the viral genome and that, often in concert with the envelope, allow for cell entry and fusion. Atomic details underlying assembly modulation are generally studied using preassembled protein complexes, while the activity of assembly modulators during assembly remains largely open and poorly understood, as necessary tools are lacking. We here use the full-length hepatitis B virus (HBV) capsid protein (Cp183) as a model to present a combination of cell-free protein synthesis and solid-state NMR as an approach which shall open the possibility to produce and analyze the formation of higher-order complexes directly on exit from the ribosome. We demonstrate that assembled capsids can be synthesized in amounts sufficient for structural studies, and show that addition of assembly modulators to the cell-free reaction produces objects similar to those obtained by addition of the compounds to preformed Cp183 capsids. These results establish the cell-free system as a tool for the study of capsid assembly modulation directly after synthesis by the ribosome, and they open the perspective of assessing the impact of natural or synthetic compounds, or even enzymes that perform post-translational modifications, on capsids structures.

Published
2019
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46. Hepatitis B virus core protein phosphorylation: Identification of the SRPK1 target sites and impact of their occupancy on RNA binding and capsid structure

Author

Heger-Stevic, Julia, Zimmermann, Peter, Lecoq, Lauriane, Böttcher, Bettina, Nassal, Michael, University of Freiburg [Freiburg], Microbiologie moléculaire et biochimie structurale / Molecular Microbiology and Structural Biochemistry (MMSB), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), and University of Würzburg = Universität Würzburg

Subjects
Molecular biology, viruses, Virus Replication, Biochemistry, Mass Spectrometry, Viral Packaging, Virions, Biology (General), Phosphorylation, Post-Translational Modification, RNA structure, Pathology and laboratory medicine, Viral Core Proteins, Proteases, Medical microbiology, Hepatitis B, Recombinant Proteins, Enzymes, Nucleic acids, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Viruses, RNA, Viral, Pathogens, Research Article, Hepatitis B virus, QH301-705.5, Protein Serine-Threonine Kinases, Viral Structure, Microbiology, Capsid, Virology, Humans, Medicine and health sciences, Virus Assembly, Viral pathogens, Organisms, Phosphatases, Biology and Life Sciences, Proteins, RC581-607, Viral Replication, Hepatitis viruses, Microbial pathogens, Macromolecular structure analysis, Mutagenesis, Site-Directed, Enzymology, RNA, Capsid Proteins, Immunologic diseases. Allergy, Serine Proteases
Abstract

Hepatitis B virus (HBV) replicates its 3 kb DNA genome through capsid-internal reverse transcription, initiated by assembly of 120 core protein (HBc) dimers around a complex of viral pregenomic (pg) RNA and polymerase. Following synthesis of relaxed circular (RC) DNA capsids can be enveloped and secreted as stable virions. Upon infection of a new cell, however, the capsid disintegrates to release the RC-DNA into the nucleus for conversion into covalently closed circular (ccc) DNA. HBc´s interactions with nucleic acids are mediated by an arginine-rich C terminal domain (CTD) with intrinsically strong non-specific RNA binding activity. Adaptation to the changing demands for nucleic acid binding during the viral life cycle is thought to involve dynamic phosphorylation / dephosphorylation events. However, neither the relevant enzymes nor their target sites in HBc are firmly established. Here we developed a bacterial coexpression system enabling access to definably phosphorylated HBc. Combining Phos-tag gel electrophoresis, mass spectrometry and mutagenesis we identified seven of the eight hydroxy amino acids in the CTD as target sites for serine-arginine rich protein kinase 1 (SRPK1); fewer sites were phosphorylated by PKA and PKC. Phosphorylation of all seven sites reduced nonspecific RNA encapsidation as drastically as deletion of the entire CTD and altered CTD surface accessibility, without major structure changes in the capsid shell. The bulk of capsids from human hepatoma cells was similarly highly, yet non-identically, phosphorylated as by SRPK1. While not proving SRPK1 as the infection-relevant HBc kinase the data suggest a mechanism whereby high-level HBc phosphorylation principally suppresses RNA binding whereas one or few strategic dephosphorylation events enable selective packaging of the pgRNA/polymerase complex. The tools developed in this study should greatly facilitate the further deciphering of the role of HBc phosphorylation in HBV infection and its evaluation as a potential new therapeutic target., Author summary The liver-pathogenic hepatitis B virus (HBV) is a small enveloped DNA virus that replicates through reverse transcription of a pregenomic (pg)RNA. This requires specific encapsidation of pgRNA and viral polymerase into a shell of 240 core protein (HBc) subunits. Capsid-internal formation of relaxed circular (RC) DNA enables the particle to leave the cell as stable virion; yet, when infecting a new cell it must release the RC-DNA for conversion into another, plasmid-like DNA that templates new viral RNAs. This up and down in nucleic acid interactions is presumably regulated by transient phosphorylation of HBc, mainly in its arginine-rich C terminal domain (CTD) which displays strong non-sequence-specific RNA binding. However, neither the phosphorylation sites nor the relevant enzymes are well defined. We developed a recombinant system to produce kinase-specific phospho-HBc species, and adapted a feasible gel assay for their separation. By mutagenesis and mass spectrometry we identified seven target sites for a major candidate kinase, SRPK1, in the CTD. As full SRPK1 phosphorylation thwarted non-specific RNA binding the comparably high phosphorylation of HBc in human cells suggests how specific pgRNA encapsidation might be achieved. Our new tool set will facilitate disentangling the role of HBc phosphorylation in HBV infection and exploiting it as potential therapeutic target.

Published
2018
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50. Strukturelle Untersuchung subviraler Partikel durch die Kombination von zellfreier Proteinherstellung mit 110 kHz MAS‐NMR‐Spektroskopie

Author

David, Guillaume, primary, Fogeron, Marie‐Laure, additional, Schledorn, Maarten, additional, Montserret, Roland, additional, Haselmann, Uta, additional, Penzel, Susanne, additional, Badillo, Aurélie, additional, Lecoq, Lauriane, additional, André, Patrice, additional, Nassal, Michael, additional, Bartenschlager, Ralf, additional, Meier, Beat H., additional, and Böckmann, Anja, additional

Published
2018
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