Crystal structures and atomic model of NADPH oxidase

Abstract

NADPH oxidases (NOXs) are the only enzymes exclusively dedicated to reactive oxygen species (ROS) generation. Dysregulation of these polytopic membrane proteins impacts the redox signaling cascades that control cell proliferation and death. We describe the atomic crystal structures of the catalytic flavin adenine dinucleotide (FAD)- and heme-binding domains of NOX5. The two domains form the core subunit that is common to all seven members of the NOX family. The domain structures were then docked in silico to provide a generic model for the NOX family. A linear arrangement of cofactors (NADPH, FAD, and two membrane-embedded heme moieties) injects electrons from the intracellular side across the membrane to a specific oxygen-binding cavity on the extracytoplasmic side. The overall spatial organization of critical interactions is revealed between the intracellular loops on the transmembrane domain and the NADPH-oxidizing dehydrogenase domain. In particular, the C terminus functions as a toggle switch, which affects access of the NADPH substrate to the enzyme. The essence of this mechanistic model is that the regulatory cues conformationally gate NADPH-binding, implicitly providing a handle for activating/deactivating the very first step in the redox chain. Such insight provides a framework to the discovery of much needed drugs that selectively target the distinct members of the NOX family and interfere with ROS signaling.

NADPH Oxidase 5 Is a Pro‐Contractile Nox Isoform and a Point of Cross‐Talk for Calcium and Redox Signaling‐Implications in Vascular Function

“…Nox5 is the only Nox isoform that has been crystalized 16. Unique to Nox5 is its long N‐terminal extension that contains 4 calcium (Ca 2+ )‐binding EF hands, which undergoes conformational change upon Ca 2+ binding 17, 18.…”

Down-regulation of NOX2 activity in phagocytes mediated by ATM-kinase dependent phosphorylation

“…The much longer NIS of NOX5 has been demonstrated to contain the EF-hand binding site that confers calcium control to NOX5 [35], highlighting the importance of protein/protein interactions in this area to control protein activity. This region is still invisible in the very recent crystal structure of a NOX5 homolog [36], confirming its probable mobility. As noted above, the NIS in general probably controls NADPH accessibility to its binding site, because many NIS substitutions in the NOX2 background affect the initial electron transfer [26, 37].…”

“…DNA sequences were verified by DNA sequencing on both strands using the Big Dye Kit (Cogenics, Grenoble, France). The wild type or mutated gp91 phox cDNA were then subcloned into the mammalian expression vector pEF-PGKneo as previously described [36]. The transfection of X-CGD PLB-985 cell line (lacking NOX2 expression and NADPH oxidase activity) [27] with the pEF-PGKneo constructs was performed by electroporation and clone selection was done by limiting dilution in 1.5 mg/ml of geneticin.…”

High‐resolution studies of hydride transfer in the ferredoxin:NADP+ reductase superfamily

“…For instance, for the NOX enzymes that play crucial roles in the production of superoxide and hydrogen peroxide for diverse biological processes, the FNR-like module is at the C-terminus of a membrane-bound flavocytochrome catalytic subunit that has been relatively difficult to study [14]. Although a NOX structure has long eluded structural biologists, a crystal structure of the FNR-like module of NOX5 from Cylindrospermum stagnale (referred to as the NADPH-dehydrogenase domain) was solved while this work was under review [30]. Sequence alignments of FNR with human NOXs aided by the C. stagnale NOX5 structure (Figure 9A) show that the NOX isozymes conserve many NADP + binding residues including the nicotinamide-interacting residues equivalent to corn root FNR Ser94, Cys274, Glu314 and Tyr316 (Phe693 in C. stagnale NOX5).…”

“…To accomplish this, we chose corn root FNR, for which crystals of the wild-type enzyme diffract to near 1 Å resolution [29]. We also argue for the general relevance of these insights for broader members of the FNR superfamily, including the NOX enzymes, which is further supported by the very recent structures of the core catalytic subunit domains of NOX5, published while this work was under review [30]. …”