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J. Biol. Chem., Vol. 282, Issue 35, 25737-25747, August 31, 2007

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Structural and Membrane Binding Analysis of the Phox Homology Domain of Bem1p

BASIS OF PHOSPHATIDYLINOSITOL 4-PHOSPHATE SPECIFICITY^*

Robert V. Stahelin^¶, Dimitrios Karathanassis^||1, Diana Murray^**, Roger L. Williams^||, and Wonhwa Cho²

From the Department of Chemistry, University of Illinois, Chicago, Illinois 60607-7061, the Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, and the ^¶Department of Chemistry and Biochemistry and the Walther Center for Cancer Research, University of Notre Dame, South Bend, Indiana 46617, the ^||Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom, and the ^**Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021

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 the diverse PI specificity of PX domains, we determined the crystal structure of the PX domain from Bem1p that has been reported to bind phosphatidylinositol 4-phosphate (PtdIns(4)P). We also measured the membrane binding properties of the PX domain and its mutants by surface plasmon resonance and monolayer techniques and calculated the electrostatic potentials for the PX domain in the absence and presence of bound PtdIns(4)P. The Bem1p PX domain contains a signature PI-binding site optimized for PtdIns(4)P binding and also harbors basic and hydrophobic residues on the membrane-binding surface. The membrane binding of the Bem1p PX domain is initiated by nonspecific electrostatic interactions between the cationic membrane-binding surface of the domain and anionic membrane surfaces, followed by the membrane penetration of hydrophobic residues. Unlike other PX domains, the Bem1p PX domain has high intrinsic membrane penetrating activity in the absence of PtdIns(4)P, suggesting that the partial membrane penetration may occur before specific PtdIns(4)P binding and last after the removal of PtdIns(4)P under certain conditions. This structural and functional study of the PtdIns(4)P-binding Bem1p PX domain provides new insight into the diverse PI specificities and membrane-binding mechanisms of PX domains.

Received for publication, April 4, 2007 , and in revised form, May 29, 2007.

The atomic coordinates and structure factors (code 2v6v) 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 Fig. 1.

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

² 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: 312-996-2183; E-mail: wcho{at}uic.edu.

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