Abstract
The superoxide-generating NADPH oxidase is converted to an active state by the assembly of a membrane-localized cytochrome b(559) with three cytosolic components: p47(phox), p67(phox), and GTPase Rac1 or Rac2. Assembly involves two sets of protein-protein interactions: among cytosolic components and among cytosolic components and cytochrome b(559) within its lipid habitat. We circumvented the need for interactions among cytosolic components by constructing a recombinant tripartite chimera (trimera) consisting of the Phox homology (PX) and Src homology 3 (SH3) domains of p47(phox), the tetratricopeptide repeat and activation domains of p67(phox), and full-length Rac1. Upon addition to phagocyte membrane, the trimera was capable of oxidase activation in vitro in the presence of an anionic amphiphile. The trimera had a higher affinity (lower EC(50)) for and formed a more stable complex (longer half-life) with cytochrome b(559) compared with the combined individual components, full-length or truncated. Supplementation of membrane with anionic but not neutral phospholipids made activation by the trimera amphiphile-independent. Mutagenesis, truncations, and domain replacements revealed that oxidase activation by the trimera was dependent on the following interactions: 1) interaction with anionic membrane phospholipids via the poly-basic stretch at the C terminus of the Rac1 segment; 2) interaction with p22(phox) via Trp(193) in the N-terminal SH3 domain of the p47(phox) segment, supplementing the electrostatic attraction; and 3) an intrachimeric bond among the p67(phox) and Rac1 segments complementary to their physical fusion. The PX domain of the p47(phox) segment and the insert domain of the Rac1 segment made only minor contributions to oxidase assembly.
Highlights
The superoxide-generating NADPH oxidase is converted to an active state by the assembly of a membrane-localized cytochrome b559 with three cytosolic components: p47phox, p67phox, and GTPase Rac1 or Rac2
We circumvented the need for interactions among cytosolic components by constructing a recombinant tripartite chimera consisting of the Phox homology (PX) and Src homology 3 (SH3) domains of p47phox, the tetratricopeptide repeat and activation domains of p67phox, and full-length Rac1
The Rationale on Which the Design of Trimeras Was Based— We have reported in the past that a recombinant chimeric protein consisting of p67phox-(1–212) fused with full-length Rac1 is capable of eliciting NADPH-dependent O2. production by preparations of phagocyte membranes in the presence of p47phox and an anionic amphiphilic activator [25, 40] and in the absence of p47phox and amphiphile when the chimera is prenylated [25, 41]
Summary
Chemicals and Reagents—The hydrolysis-resistant GTP analog GMPPNP (tetralithium salt, 83%, Ͻ0.2% GTP) was purchased from Roche Applied Science. Full-length p67phox, p67phox-(1–212), and p47phox-(1–286) were expressed as glutathione S-transferase (GST) fusion proteins and purified by batch affinity chromatography on glutathione-agarose (Sigma), followed by thrombin cleavage [44], as described previously [45]. The induced cells were disrupted by lysozyme treatment and by sonication in buffer consisting of 50 mM sodium phosphate, pH 7.5, and 300 mM NaCl supplemented with Complete EDTA-free protease inhibitor as described for GST-fused chimeras. The phospholipids were added to the solubilized macrophage membranes at a constant ratio of 4 volumes of phospholipid (5 mM) to 1 volume of membrane (at a concentration equivalent to 1.2 M cytochrome b559 heme) unless mentioned otherwise In another set of experiments, membranes were supplemented with DOPG and either PtdIns[3,4]P2 or PtdIns[4,5]P2. Curve Plotting—Plotting of dose-response curves and calculation of Vmax and EC50 values were performed using GraphPad Prism Version 4.03
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