Binding of the PX domain of p47(phox) to phosphatidylinositol 3,4-bisphosphate and phosphatidic acid is masked by an intramolecular interaction

p47(phox) is a key cytosolic subunit required for activation of phagocyte NADPH oxidase. The X-ray structure of the p47(phox) PX domain revealed two distinct basic pockets on the membrane-binding surface, each occupied by a sulfate. These two pockets have different specificities: one preferentially...

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Bibliographic Details
Published in:The EMBO journal 2002-10, Vol.21 (19), p.5057
Main Authors: Karathanassis, Dimitrios, Stahelin, Robert V, Bravo, Jerónimo, Perisic, Olga, Pacold, Christine M, Cho, Wonhwa, Williams, Roger L
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino Acid Substitution
Binding Sites
Cell Membrane - physiology
Cell Membrane - ultrastructure
Cloning, Molecular
Humans
Kinetics
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
NADPH Oxidases - chemistry
NADPH Oxidases - metabolism
Phosphatidic Acids - chemistry
Phosphatidic Acids - metabolism
Phosphatidylinositol Phosphates - chemistry
Phosphatidylinositol Phosphates - metabolism
Phosphatidylinositols - chemistry
Phosphatidylinositols - metabolism
Phosphoproteins - chemistry
Phosphoproteins - metabolism
Polymerase Chain Reaction
Protein Conformation
Protein Structure, Secondary
Protein Subunits
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Sequence Alignment
Sequence Homology, Amino Acid
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Summary:p47(phox) is a key cytosolic subunit required for activation of phagocyte NADPH oxidase. The X-ray structure of the p47(phox) PX domain revealed two distinct basic pockets on the membrane-binding surface, each occupied by a sulfate. These two pockets have different specificities: one preferentially binds phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)] and is analogous to the phophatidylinositol 3-phosphate (PtdIns3P)-binding pocket of p40(phox), while the other binds anionic phospholipids such as phosphatidic acid (PtdOH) or phosphatidylserine. The preference of this second site for PtdOH may be related to previously observed activation of NADPH oxidase by PtdOH. Simultaneous occupancy of the two phospholipid-binding pockets radically increases membrane affinity. Strikingly, measurements for full-length p47(phox) show that membrane interaction by the PX domain is masked by an intramolecular association with the C-terminal SH3 domain (C-SH3). Either a site-specific mutation in C-SH3 (W263R) or a mimic of the phosphorylated form of p47(phox) [Ser(303, 304, 328, 359, 370)Glu] cause a transition from a closed to an open conformation that binds membranes with a greater affinity than the isolated PX domain.
ISSN:0261-4189
1460-2075
DOI:10.1093/emboj/cdf519