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Functional control of a 0.5 MDa TET aminopeptidase by a flexible loop revealed by MAS NMR

By Diego F. Gauto, Pavel Macek, Duccio Malinverni, Hugo Fraga, Matteo Paloni, Iva Sučec, Audrey Hessel, Juan Pablo Bustamante, Alessandro Barducci, Paul Schanda

Posted 30 Jun 2021
bioRxiv DOI: 10.1101/2021.06.29.450317

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 [A] 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 molecu- lar 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 addition- ally 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.

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