The reactive oxygen species (ROS)-producing enzyme NADPH oxidase (NOX) was first identified in the membrane of phagocytic cells. For many years, its only known role was in immune defense, where its ROS production leads to the destruction of pathogens by the immune cells. NOX from phagocytes catalyzes, via one-electron trans-membrane transfer to molecular oxygen, the production of the superoxide anion. Over the years, six human homologs of the catalytic subunit of the phagocyte NADPH oxidase were found: NOX1, NOX3, NOX4, NOX5, DUOX1, and DUOX2. Together with the NOX2/gp91^phox component present in the phagocyte NADPH oxidase assembly itself, the homologs are now referred to as the NOX family of NADPH oxidases. NOX are complex multidomain proteins with varying requirements for assembly with combinations of other proteins for activity. The recent structural insights acquired on both prokaryotic and eukaryotic NOX open new perspectives for the understanding of the molecular mechanisms inherent to NOX regulation and ROS production (superoxide or hydrogen peroxide). This new structural information will certainly inform new investigations of human disease. As specialized ROS producers, NOX enzymes participate in numerous crucial physiological processes, including host defense, the post-translational processing of proteins, cellular signaling, regulation of gene expression, and cell differentiation. These diversities of physiological context will be discussed in this review. We also discuss NOX misregulation, which can contribute to a wide range of severe pathologies, such as atherosclerosis, hypertension, diabetic nephropathy, lung fibrosis, cancer, or neurodegenerative diseases, giving this family of membrane proteins a strong therapeutic interest.

An optimised, economical cell-free protocol for producing perdeuterated, methyl-labelled samples of the ClpXP machine, with separate labelling of the ClpX and ClpP components, enables the acquisition of high-resolution 2D methyl-TROSY NMR spectra.

NADPH oxidases (NOX) are transmembrane proteins that professionally produce reactive oxygen species (ROS) and are distributed widely in both eukaryotes and prokaryotes. Eukaryotes use the ROS products for innate immune defense and signaling; the seven human isoforms of NOX participate in critical physiological and pathophysiological processes. Recently solved structures of two human NOX isoforms provide much new information, but do not fully elucidate controls on the electron transfer pathway from NAD(P)H substrate through FAD and heme cofactors to the final ROS product. SpNOX, a bacterial NOX homolog from Streptococcus pneumoniae , shows robust constitutive activity in detergent solution, making it a good prototype for exploring electron transfer in the NOX family. Here we present crystal structures of wildtype and mutant full-length and dehydrogenase (DH) domain-only constructs of SpNOX. The isolated DH domain acts as a flavin reductase, and both DH and full-length constructs use either NADPH or NADH as substrate. Our data supports hydride transfer from NAD(P)H to FAD as the rate limiting step in electron transfer. Using the DH domain, we demonstrate the role of F397 in allowing access of nicotinamide to the flavin isoalloxazine, while in the full-length construct, we used mutants and flavin analogs to confirm the contribution of both domains to flavin binding observed in the structure. Comparison with homologous enzymes suggests distal heme access may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity of a given NOX family member. Based on this comparison, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2’s requirement for activation.

AAA+ ATPases are ring-shaped hexameric protein complexes that operate as elaborate macromolecular motors, driving a variety of ATP-dependent cellular processes. AAA+ ATPases undergo large-scale conformational changes that lead to the conversion of chemical energy from ATP into mechanical work to perform a wide range of functions, such as unfolding and translocation of the protein substrate inside a proteolysis chamber of an AAA+-associated protease. Despite extensive biochemical studies on these macromolecular assemblies, the mechanism of substrate unfolding and degradation has long remained elusive. Indeed, until recently, structural characterization of AAA+ protease complexes remained hampered by the size and complexity of the machinery, harboring multiple protein subunits acting together to process proteins to be degraded. Additionally, the major structural rearrangements involved in the mechanism of this complex represent a crucial challenge for structural biology. Here, we report the main advances in deciphering molecular details of the proteolytic reaction performed by AAA+ proteases, based on the remarkable progress in structural biology techniques. Particular emphasis is placed on the latest findings from high-resolution structural analysis of the ClpXP proteolytic complex, using crystallographic and cryo-EM investigations. In addition, this review presents some additional dynamic information obtained using solution-state NMR. This information provides molecular details that help to explain the protein degradation process by such molecular machines.

NADPH oxidases (NOX) are transmembrane proteins that professionally produce reactive oxygen species (ROS) and are distributed widely in both eukaryotes and prokaryotes. Eukaryotes use the ROS products for innate immune defense and signaling; the seven human isoforms of NOX participate in critical physiological and pathophysiological processes. Recently solved structures of two human NOX isoforms provide much new information, but do not fully elucidate controls on the electron transfer pathway from NAD(P)H substrate through FAD and heme cofactors to the final ROS product. SpNOX, a bacterial NOX homolog from Streptococcus pneumoniae, shows robust constitutive activity in detergent solution, making it a good prototype for exploring electron transfer in the NOX family. Here we present crystal structures of wildtype and mutant full-length and dehydrogenase (DH) domain-only constructs of SpNOX. The isolated DH domain acts as a flavin reductase, and both DH and full-length constructs use either NADPH or NADH as substrate. Our data supports hydride transfer from NAD(P)H to FAD as the rate limiting step in electron transfer. Using the DH domain, we demonstrate the role of F397 in allowing access of nicotinamide to the flavin isoalloxazine, while in the full-length construct, we used mutants and flavin analogs to confirm the contribution of both domains to flavin binding observed in the structure. Comparison with homologous enzymes suggests distal heme access may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity of a given NOX family member. Based on this comparison, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2’s requirement for activation.

En tant qu’enzymes spécialisées dans la production d’espèces réactives de l’oxygène (ROS), le rôle des NADPH oxydases (NOX) s’étend à de nombreux processus physiologiques majeurs, et la dérégulation de celles-ci peut conduire à une grande variété de pathologies sévères, telles que le diabète ou les maladies cardiovasculaires et neurodégénératives, constituant ainsi une cible primordiale pour le développement de solutions thérapeutiques spécifiquement adaptées. Malgré la découverte récente d’homologues procaryotes de NOX, facilitant la production de ces protéines en quantités compatibles pour les études mécanistiques, et constituant ainsi des modèles favorisant l’étude fonctionnelle et structurale de la famille des NADPH oxydases, la résolution d’une structure haute résolution sous forme entière est toujours demeurée sans solution.Ainsi, à travers l’utilisation d’une approche interdisciplinaire, ce projet a pour but de relever les enjeux majeurs associés à l’obtention de cristaux dont la qualité de diffraction permettrait la résolution de la première structure d’un homologue de NADPH oxydase sous forme entière issu de Streptococcus pneumoniae (SpNox), complexifiée par la présence de larges zones hydrophobes intrinsèques au rôle de transfert d’électrons à travers les membranes de ces protéines, et pouvant altérer la formation et l’intégrité des contacts cristallins.Des conditions optimales de détergents et de tampons au sein de laquelle l’enzyme combine une importante activité associée à une haute thermostabilité ont été identifiées par criblage systématique des conditions de sels et pH pour la purification de l’enzyme et ainsi optimiser la préservation de l’intégrité de la protéine afin de favoriser la qualité des cristaux obtenus. De plus, à travers l’implémentation d’étapes conjointes de criblages ainsi que d’affinements automatiques et manuels extensifs, un effort intense a ainsi été initié afin d’optimiser le processus de cristallogenèse et des avancées majeures ont pu être réalisées concernant la cristallisation de SpNox permettant d’améliorer la diffraction des cristaux initiaux de 11Å à 4.3Å.En parallèle, une caractérisation basse résolution, réalisée par diffusion des neutrons aux petits angles, a permis de déterminer une enveloppe globale de la protéine. Le volume de cette enveloppe, plus large que le modèle d’homologie de la protéine sous forme entière reconstituée par l’ancrage des deux domaines modélisés indépendamment, suggère l’existence d’une flexibilité importante de SpNox dans la région charnière entre les domaines TM et DH, fournissant ainsi des informations essentielles et apportant une explication aux difficultés rencontrées pour abaisser la limite de résolution des cristaux.Afin de s‘affranchir de la flexibilité liée à la présence de deux domaines structuralement indépendants au sein de la protéine, une stratégie complémentaire, impliquant la caractérisation structurale de chacun des domaines dissociés de SpNox a été initiée en caractérisant indépendamment avec succès les domaines transmembranaire (TM) et déshydrogénase (DH) de SpNox. Des développements considérables ont été réalisés grâce à l’obtention de cristaux du domaine déshydrogénase, dont la qualité a permis la collecte de données selon des limites de diffraction atteignant 1.8Å permettant ainsi d’accéder à des informations à une résolution atomique, autorisant la visualisation de détails structuraux au sein de ce domaine, qui, en association avec le modèle d’homologie du TM obtenu par une approche bioinformatique, peut servir à guider certaines études fonctionnelles.