10779/crick.12571178.v1 Elisa Millana Fañanás Elisa Millana Fañanás Sofia Todesca Sofia Todesca Alessandro Sicorello Alessandro Sicorello Laura Masino Laura Masino Petr Pompach Petr Pompach Francesca Magnani Francesca Magnani Annalisa Pastore Annalisa Pastore Andrea Mattevi Andrea Mattevi On the mechanism of calcium-dependent activation of NADPH oxidase 5 (NOX5). The Francis Crick Institute 2020 EF-hands NMR calcium activation enzyme structure SB NMR-ack Biochemistry & Molecular Biology 0601 Biochemistry and Cell Biology 1101 Medical Biochemistry and Metabolomics 0304 Medicinal and Biomolecular Chemistry 2020-07-01 11:55:05 Journal contribution https://crick.figshare.com/articles/journal_contribution/On_the_mechanism_of_calcium-dependent_activation_of_NADPH_oxidase_5_NOX5_/12571178 It is now accepted that reactive oxygen species (ROS) are not only dangerous oxidative agents but also chemical mediators of the redox cell signaling and innate immune response. A central role in ROS-controlled production is played by the NADPH oxidases (NOXs), a group of seven membrane-bound enzymes (NOX1-5 and DUOX1-2) whose unique function is to produce ROS. Here, we describe the regulation of NOX5, a widespread family member present in cyanobacteria, protists, plants, fungi, and the animal kingdom. We show that the calmodulin-like regulatory EF-domain of NOX5 is partially unfolded and detached from the rest of the protein in the absence of calcium. In the presence of calcium, the C-terminal lobe of the EF-domain acquires an ordered and more compact structure that enables its binding to the enzyme dehydrogenase (DH) domain. Our spectroscopic and mutagenesis studies further identified a set of conserved aspartate residues in the DH domain that are essential for NOX5 activation. Altogether, our work shows that calcium induces an unfolded-to-folded transition of the EF-domain that promotes direct interaction with a conserved regulatory region, resulting in NOX5 activation.