Gauto, Diego F.; Macek, Pavel; Malinverni, Duccio; Fraga, Hugo; Paloni, Matteo; Sučec, Iva; Hessel, Audrey; Bustamante, Juan Pablo; Barducci, Alessandro; Schanda, PaulISTA
Large oligomeric enzymes control a myriad of cellular processes, from protein synthesis and degradation to metabolism. The 0.5 MDa large TET2 aminopeptidase, a prototypical protease important for cellular homeostasis, degrades peptides within a ca. 60 Å wide tetrahedral chamber with four lateral openings. The mechanisms of substrate trafficking and processing remain debated. Here, we integrate magic-angle spinning (MAS) NMR, mutagenesis, co-evolution analysis and molecular dynamics simulations and reveal that a loop in the catalytic chamber is a key element for enzymatic function. The loop is able to stabilize ligands in the active site and may additionally have a direct role in activating the catalytic water molecule whereby a conserved histidine plays a key role. Our data provide a strong case for the functional importance of highly dynamic - and often overlooked - parts of an enzyme, and the potential of MAS NMR to investigate their dynamics at atomic resolution.
We are grateful to Bernhard Brutscher, Alicia Vallet, and Adrien Favier for excellent NMR platform operation and management. The plasmid coding for TET2 was kindly provided by Bruno Franzetti and Jerome Boisbouvier (IBS Grenoble). We thank Anne-Marie Villard and the RoBioMol platform for preparing the loop deletion construct. The RoBioMol platform is part of the Grenoble Instruct-ERIC center (ISBG; UAR 3518 CNRS-CEAUGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005-02) and GRAL (ANR-10-LABX-49-01), financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBHEUR-GS (ANR-17-EURE-0003). This work was supported by the European Research Council (StG-2012-311318-ProtDyn2Function to P. S.) and the French Agence Nationale de la Recherche (ANR), under grant ANR-14-ACHN-0016 (M.P. and A.B.).
Gauto DF, Macek P, Malinverni D, et al. Functional control of a 0.5 MDa TET aminopeptidase by a flexible loop revealed by MAS NMR. Nature Communications. 2022;13. doi:10.1038/s41467-022-29423-0
Gauto, D. F., Macek, P., Malinverni, D., Fraga, H., Paloni, M., Sučec, I., … Schanda, P. (2022). Functional control of a 0.5 MDa TET aminopeptidase by a flexible loop revealed by MAS NMR. Nature Communications. Springer Nature. https://doi.org/10.1038/s41467-022-29423-0
Gauto, Diego F., Pavel Macek, Duccio Malinverni, Hugo Fraga, Matteo Paloni, Iva Sučec, Audrey Hessel, Juan Pablo Bustamante, Alessandro Barducci, and Paul Schanda. “Functional Control of a 0.5 MDa TET Aminopeptidase by a Flexible Loop Revealed by MAS NMR.” Nature Communications. Springer Nature, 2022. https://doi.org/10.1038/s41467-022-29423-0.
D. F. Gauto et al., “Functional control of a 0.5 MDa TET aminopeptidase by a flexible loop revealed by MAS NMR,” Nature Communications, vol. 13. Springer Nature, 2022.
Gauto DF, Macek P, Malinverni D, Fraga H, Paloni M, Sučec I, Hessel A, Bustamante JP, Barducci A, Schanda P. 2022. Functional control of a 0.5 MDa TET aminopeptidase by a flexible loop revealed by MAS NMR. Nature Communications. 13, 1927.
Gauto, Diego F., et al. “Functional Control of a 0.5 MDa TET Aminopeptidase by a Flexible Loop Revealed by MAS NMR.” Nature Communications, vol. 13, 1927, Springer Nature, 2022, doi:10.1038/s41467-022-29423-0.
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