Abstract
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.
Highlights
Radical oxygen species (ROS) have a dual role: They are the cytotoxic molecules at the heart of oxidative stress but are critical chemical agents in immune response as well as in signaling pathways that mediate cell growth, differentiation, and death [1,2,3]
Size-exclusion chromatography showed that the EF-domain of hNOX5 has a different behavior in the presence and in the absence of calcium (Fig. 1B): The calcium-free domain eluted at earlier volumes than the calcium-bound protein, The FEBS Journal 287 (2020) 2486–2503 a 2019 The Authors
Since some canonical EF-hands can be activated by Mg2+, whose concentration in cells is higher than that of calcium (0.5–5 mM) [21], we explored the specificity of the hNOX5 EF-domain for calcium. 1H-15N HSQC spectra recorded in the presence/absence of an excess of Mg2+ showed only minor differences and no conversion to the holo-form (Fig. 2D)
Summary
Radical oxygen species (ROS) have a dual role: They are the cytotoxic molecules at the heart of oxidative stress but are critical chemical agents in immune response as well as in signaling pathways that mediate cell growth, differentiation, and death [1,2,3]. Main players in the ROS-mediated processes are members of the NADPH oxidase (NOX) family. NOX enzymes share a catalytic core formed by six transmembrane helices, with two noncovalent heme molecules, followed by a C-terminal dehydrogenase (DH) domain that binds NADPH and FAD. Electrons donated by cytosolic NADPH are sequentially transferred to FAD, heme, and lastly to an O2 molecule to produce O2- radicals and/or H2O2 at the opposite side of the cell membrane [4]. The distinction among NOX enzymes stems from their specific regulatory
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