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J. Biol. Chem., Vol. 281, Issue 51, 39396-39406, December 22, 2006

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Structural and Membrane Binding Analysis of the Phox Homology Domain of Phosphoinositide 3-Kinase-C2^*

Robert V. Stahelin, Dimitrios Karathanassis¹, Karol S. Bruzik^¶, Michael D. Waterfield^||, Jerónimo Bravo², Roger L. Williams, and Wonhwa Cho³

From the Departments of Chemistry and ^¶Medicinal Chemistry and Pharmacognosy, University of Illinois, Chicago, Illinois 60607, MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom, and ^||Ludwig Institute of Cancer Research, University College, London W1P 8BT, United Kingdom

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 C2 (PI3K-C2), which binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂). 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)P₂ 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)P₂. Furthermore, the PX domain displayed significantly higher PtdIns(4,5)P₂ membrane affinity and specificity when compared with the PI3K-C2 C2 domain, demonstrating that high affinity PtdIns(4,5)P₂ binding was facilitated by the PX domain in full-length PI3K-C2. Together, these studies provide new structural insight into the diverse PI specificities of PX domains and elucidate the mechanism by which the PI3K-C2 PX domain interacts with PtdIns(4,5)P₂-containing membranes and thereby mediates the membrane recruitment of PI3K-C2.

Received for publication, July 25, 2006 , and in revised form, October 10, 2006.

The atomic coordinates and structure factors (code 2iwl) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health Grant GM68849 (to W. C.) and by the Medical Research Council (to R. L. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Methods and supplemental Fig. S1.

¹ Present address: Research Center for Biomaterials S.A. 15, 16562 Glyfada-Athens, Greece.

² Present address: Centro Nacional de Investigaciones Oncológicas, Melchor Fernández Almagro 3, E-28029 Madrid, Spain.

³ To whom correspondence should be addressed: Dept. of Chemistry (M/C 111), University of Illinois, 845 West Taylor St., Chicago, IL 60607-7061. Tel.: 312-996-4883; Fax: 312-996-0431; E-mail: wcho{at}uic.edu.

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