NADPH oxidase activator p67^phox behaves in solution as a multidomain protein with semi-flexible linkers

Abstract

The NADPH oxidase complex is involved in the destruction of phagocytosed pathogens through the production of reactive oxygen species. This activatable complex consists of a membranous heterodimeric flavocytochrome b, a small G-protein Rac1/Rac2 and cytosolic factors, p47^phox, p67^phox and p40^phox. p67^phox, due to its modular structure, is the NADPH oxidase component for which global structure information is most scarce despite its mandatory role in activation and its central position in the whole complex organization. Indeed, p67^phox is the only factor establishing interaction with all others. In this study, we report the SAXS analysis of p67^phox. Our data reveals that p67^phox behaves as a multidomain protein with semi-flexible linkers. On the one hand, it appears to be a very elongated molecule with its various domains organized as beads on a string. Linkers are predicted to be partially or mainly unstructured and features of our experimental data do point towards inter-domain flexibility. On the other hand, our work also suggests that the protein is not as extended as unstructured linkers could allow, thereby implying the existence of intra-molecular interactions within p67^phox. We suggest that the dual character of p67^phox conformation in solution is central to ensure the numerous interactions to be accommodated.

Introduction

Neutrophils play a key role in host defence against invading microorganisms. They possess a multi-component system, the NADPH oxidase, involved in the destruction of phagocytosed pathogens through the production of superoxide anion O₂^•− and other microbicidal derivatives upon activation (Lambeth, 2004, Vignais, 2002). This complex consists of a membrane inserted heterodimeric flavocytochrome b, a small G-protein Rac1/Rac2 and cytosolic factors, p47^phox, p67^phox and p40^phox. The importance of this complex in innate immunity is underlined by the occurrence of chronic granulomatous diseases (CGD) upon mutation(s) within one of its components. The neutrophil NADPH oxidase complex, initially considered as a specific activity of phagocytic immune cells, became the prototype of a family of enzymes recently discovered and widely spread in many different cell types and tissues and associated to numerous physiological functions (Bedard and Krause, 2007, Nauseef, 2008).

Reactive oxygen species production is tightly regulated to avoid self-tissue injury and is under the strict dependence of the assembly of cytosolic factors with the membrane redox component. These cytosolic factors are suggested to form a ternary cytosolic complex in the cytoplasm. The activation process is initiated through multiple phosphorylations of the cytosolic factors (El Benna et al., 1994, Inanami et al., 1998, Johnson et al., 1998). This causes conformational changes within the ternary complex, and concomitant translocation, with Rac-GTP, to the membrane embedded flavocytochrome b₅₅₈. The molecular mechanisms triggered by these sets of phosphorylation events have long been the object of numerous investigations. Indeed, a better understanding of the molecular details of the activation process is a prerequisite to the design of a strategy aiming at a control of NADPH oxidase activity. Despite strong progress made over the years, and notably regarding the structural characterization of the various components, many aspects and details of the molecular mechanism still need to be elucidated. Among the three cytosolic factors, only p47^phox and p67^phox are known to be essential for activation since they are related to CGD pathology, through inactivating mutations, in contrast to p40^phox. p47^phox is the main target of the phosphorylation reactions and is central in the assembly process as it drives the translocation of other cytosolic factors to the membrane for assembly (Heyworth et al., 1991). Indeed, p47^phox is defined as the complex “organizer” protein. As for p67^phox, it is defined as the “activator” component of the NADPH oxidase, together with Rac, since it is essential to induce electron-transfer in flavocytochrome b₅₅₈ (Li et al., 2007, Nisimoto et al., 1999). The sole requirement for p67^phox and Rac1/2 soluble components, in an in vitro cell free system, to promote a NADPH oxidase activity illustrates this point (Dagher and Pick, 2007).

The modular nature of the cytosolic factors p47^phox and p67^phox has made them reluctant to crystallization and prevented the X-ray structure determination of both full-length proteins, in contrast to p40^phox (Honbou et al., 2006, Honbou et al., 2007). Structural studies of these proteins were therefore focused on the characterization of their isolated modules (Grizot et al., 2001a, Groemping et al., 2003, Hiroaki et al., 2001, Kami et al., 2002, Karathanassis et al., 2002, Ogura et al., 2006, Royant et al., 2002, Wilson et al., 2003). The resulting structures yielded information of great relevance to our understanding of properties associated to each particular module: the interaction of PX domains with lipids (Bravo et al., 2001, Karathanassis et al., 2002, Málková et al., 2006, Stahelin et al., 2003), the molecular explanation of several CGD mutations (Grizot et al., 2001a), a structural view of interactions between cytosolic factors (Kami et al., 2002, Massenet et al., 2005, Ogura et al., 2006, Wilson et al., 2003) and also some insight on the effects of phosphorylations (Groemping et al., 2003, Yuzawa et al., 2004). However, limits in the interpretation have been met when considering the global organization of the modules in full-length proteins and their complexes. Structural information on entire components, even at low resolution, appeared as being crucial to characterize global protein organization and dynamics during the activation process. As an illustration, attempts to described the auto-inhibited organization of p47^phox in the resting state based only on isolated domain structures gave irreconcilable results. On the basis of a SAXS study of full-length p47^phox, we could propose an alternative auto-inhibited organization compatible with known biochemical data (Durand et al., 2006). Several studies have since furthered our understanding of the autoinhibitory state of p47^phox (Marcoux et al., 2009, Shen et al., 2008, Ueyama et al., 2008). Apart from these studies on entire p47^phox, very few studies approaching organization and/or molecular structure of entire components of the NADPH oxidase complex have met with success with the remarkable exception of the already mentioned structure determination of full-length p40^phox (Grizot et al., 2001b, Grizot et al., 2001c, Honbou et al., 2007).

In that context, p67^phox is probably the NADPH oxidase component for which global structure information is most scarce. This is surprising regarding its mandatory role in NADPH oxidase activation and its central position in the whole complex organization. Indeed, among the various components (p47^phox, p40^phox, Rac, and the flavocytochrome b₅₅₈), p67^phox is the only factor establishing interaction with all others (Fig. 1). It comprises, from its N-terminal to its C-terminal end, several successive domains: a TPR domain involved in Rac1/2 interaction, followed by an activation domain responsible for the activation of the flavocytochrome b₅₅₈, a first SH3 domain of unknown function, a PB1 domain that interacts with p40^phox and the terminal SH3 domain that allows membrane translocation, upon activation, through an interaction with p47^phox. In addition, phosphorylation events on p67^phox during NADPH oxidase activation have been reported, but the mechanism by which they could regulate the NADPH oxidase activation remains obscure (El Benna et al., 1997, Dusi and Rossi, 1993, Forbes et al., 1999). Finally, new potential interacting partners for p67^phox have been recently suggested, raising new questions about the organization and regulation of the NADPH oxidase complex (Marty et al., 2006, Ming et al., 2007). In that context, any information regarding p67^phox global structure, albeit at low resolution, could help understand such a complex organization around this multi-interacting platform and may shed some light on unexpected element of its regulation.

In this study, we report the SAXS analysis of p67^phox in solution. Our data reveals that p67^phox is a very elongated molecule with its various domains organized as beads on a string. All linkers are predicted to be partially or mainly unstructured. However, even if p67^phox adopts a large number of conformers in solution, our data suggest that intra-molecular interactions within p67^phox may well be present.