title: Structural and membrane binding analysis of the Phox homology domain of phosphoinositide 3-kinase-C2alpha.
abstract: Phox homology (PX) domains, which have been identified in a variety of proteins involved in cell signaling and membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of diverse PI specificities of PX domains, we determined the crystal structure of the PX domain from phosphoinositide 3-kinase C2alpha (PI3K-C2alpha), which binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). To delineate the mechanism by which this PX domain interacts with membranes, we measured the membrane binding of the wild type domain and mutants by surface plasmon resonance and monolayer techniques. This PX domain contains a signature PI-binding site that is optimized for PtdIns(4,5)P(2) binding. The membrane binding of the PX domain is initiated by nonspecific electrostatic interactions followed by the membrane penetration of hydrophobic residues. Membrane penetration is specifically enhanced by PtdIns(4,5)P(2). Furthermore, the PX domain displayed significantly higher PtdIns(4,5)P(2) membrane affinity and specificity when compared with the PI3K-C2alpha C2 domain, demonstrating that high affinity PtdIns(4,5)P(2) binding was facilitated by the PX domain in full-length PI3K-C2alpha. Together, these studies provide new structural insight into the diverse PI specificities of PX domains and elucidate the mechanism by which the PI3K-C2alpha PX domain interacts with PtdIns(4,5)P(2)-containing membranes and thereby mediates the membrane recruitment of PI3K-C2alpha.
authors.affiliation: Departments of Chemistry and Medicinal Chemistry and Pharmacognosy, University of Illinois, Chicago, Illinois 60607, USA.
authors.forename: Robert V
authors.initials: RV
authors.lastname: Stahelin
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Karathanassis
authors.forename: Karol S
authors.initials: KS
authors.lastname: Bruzik
authors.forename: Michael D
authors.initials: MD
authors.lastname: Waterfield
authors.forename: Jerónimo
authors.initials: J
authors.lastname: Bravo
authors.forename: Roger L
authors.initials: RL
authors.lastname: Williams
authors.forename: Wonhwa
authors.initials: W
authors.lastname: Cho
mesh_terms: D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D058541:Class II Phosphatidylinositol 3-Kinases; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008958:Models, Molecular; D016296:Mutagenesis; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism / Q000502:physiology*; D010716:Phosphatidylinositols / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D055672:Static Electricity; D020349:Surface Plasmon Resonance
chemical_list: D058541:Class II Phosphatidylinositol 3-Kinases; D008055:Lipids; D019869:Phosphatidylinositol 3-Kinases; D010716:Phosphatidylinositols