Tripartite chimeras comprising functional domains derived from the cytosolic NADPH oxidase components p47phox, p67phox, and Rac1 elicit activator-independent superoxide production by phagocyte membranes: An essential role for anionic membrane phospholipids

Yevgeny Berdichevsky, Ariel Mizrahi, Yelena Ugolev, Shahar Molshanski-Mor, Edgar Pick*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

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. 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 p47phox, the tetratricopeptide repeat and activation domains of p67phox, 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 b559 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 p22phox via Trp193 in the N-terminal SH3 domain of the p47phox segment, supplementing the electrostatic attraction; and 3) an intrachimeric bond among the p67phox and Rac1 segments complementary to their physical fusion. The PX domain of the p47phox segment and the insert domain of the Rac1 segment made only minor contributions to oxidase assembly.

Original languageEnglish
Pages (from-to)22122-22139
Number of pages18
JournalJournal of Biological Chemistry
Volume282
Issue number30
DOIs
StatePublished - 27 Jul 2007

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