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J. Biol. Chem., Vol. 282, Issue 42, 30949-30959, October 19, 2007

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 282/42/30949    most recent M705499200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Strahl, T. Articles by Ames, J. B. Search for Related Content PubMed PubMed Citation Articles by Strahl, T. Articles by Ames, J. B. Social Bookmarking                      What's this?

Structural Insights into Activation of Phosphatidylinositol 4-Kinase (Pik1) by Yeast Frequenin (Frq1)^*

Thomas Strahl, Inken G. Huttner, Jacqueline D. Lusin, Masanori Osawa¹, David King, Jeremy Thorner, and James B. Ames²

From the Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720-3202 and the Department of Chemistry, University of California, Davis, California 95616

Yeast frequenin (Frq1), a small N-myristoylated EF-hand protein, activates phosphatidylinositol 4-kinase Pik1. The NMR structure of Ca²⁺-bound Frq1 complexed to an N-terminal Pik1 fragment (residues 121-174) was determined. The Frq1 main chain is similar to that in free Frq1 and related proteins in the same branch of the calmodulin superfamily. The myristoyl group and first eight residues of Frq1 are solvent-exposed, and Ca²⁺ binds the second, third, and fourth EF-hands, which associate to create a groove with two pockets. The Pik1 peptide forms two helices (125-135 and 156-169) connected by a 20-residue loop. Side chains in the Pik1 N-terminal helix (Val-127, Ala-128, Val-131, Leu-132, and Leu-135) interact with solvent-exposed residues in the Frq1 C-terminal pocket (Leu-101, Trp-103, Val-125, Leu-138, Ile-152, and Leu-155); side chains in the Pik1 C-terminal helix (Ala-157, Ala-159, Leu-160, Val-161, Met-165, and Met-167) contact solvent-exposed residues in the Frq1 N-terminal pocket (Trp-30, Phe-34, Phe-48, Ile-51, Tyr-52, Phe-55, Phe-85, and Leu-89). This defined complex confirms that residues in Pik1 pinpointed as necessary for Frq1 binding by site-directed mutagenesis are indeed sufficient for binding. Removal of the Pik1 N-terminal region (residues 8-760) from its catalytic domain (residues 792-1066) abolishes lipid kinase activity, inconsistent with Frq1 binding simply relieving an autoinhibitory constraint. Deletion of the lipid kinase unique motif (residues 35-110) also eliminates Pik1 activity. In the complex, binding of Ca²⁺-bound Frq1 forces the Pik1 chain into a U-turn. Frq1 may activate Pik1 by facilitating membrane targeting via the exposed N-myristoyl group and by imposing a structural transition that promotes association of the lipid kinase unique motif with the kinase domain.

Received for publication, July 5, 2007 , and in revised form, August 14, 2007.

The atomic coordinates and structure factors (code 2JU0) 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 Grants EY012347, NS045909 (to J. B. A.), and GM21841 (to J. T.) from the National Institutes of Health. 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 Figs. 1 and 2.

¹ Present address: Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan.

² To whom correspondence should be addressed: Dept. of Chemistry, One Shields Ave., University of California, Davis, CA 95616. Tel.: 530-752-6358; Fax: 530-752-8995; E-mail: ames{at}chem.ucdavis.edu.

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