Membrane binding mechanisms of the PX domains of NADPH oxidase p40phox and p47phox

Robert V. Stahelin, Aura Burian, Karol S. Bruzik, Diana Murray, Wonhwa Cho

Research output: Contribution to journalArticle

115 Scopus citations

Abstract

Phox (PX) domains are phosphoinositide (PI)-binding domains with broad PI specificity. Two cytosolic components of NADPH oxidase, p40phox and p47phox, contain PX domains. The PX domain of p40phox specifically binds phosphatidylinositol 3-phosphate, whereas the PX domain of p47phox has two lipid binding sites, one specific for phosphatidylinositol 3,4-bisphosphate and the other with affinity for phosphatidic acid or phosphatidylserine. To delineate the mechanisms by which these PX domains interact with PI-containing membranes, we measured the membrane binding of these domains and respective mutants by surface plasmon resonance and monolayer techniques and also calculated the electrostatic potentials of the domains as a function of PI binding. Results indicate that membrane binding of both PX domains is initiated by nonspecific electrostatic interactions, which is followed by the membrane penetration of hydrophobic residues. The membrane penetration of the p40phox PX domain is induced by phosphatidylinositol 3-phosphate, whereas that of the p47phox, PX domain is triggered by both phosphatidylinositol 3,4-bisphosphate and phosphatidic acid (or phosphatidylserine). Studies of enhanced green fluorescent protein-fused PX domains in HEK293 cells indicate that this specific membrane penetration is also important for subcellular localization of the two PX domains. Further studies on the full-length p40phox and p47phox, proteins showed that an intramolecular interaction between the C-terminal Src homology 3 domain and the PX domain prevents the nonspecific monolayer penetration of p47phox, whereas such an interaction is absent in p40phox.

Original languageEnglish (US)
Pages (from-to)14469-14479
Number of pages11
JournalJournal of Biological Chemistry
Volume278
Issue number16
DOIs
StatePublished - Apr 18 2003
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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