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J. Biol. Chem., Vol. 279, Issue 9, 8441-8451, February 27, 2004

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NMR Solution Structure of the Focal Adhesion Targeting Domain of Focal Adhesion Kinase in Complex with a Paxillin LD Peptide

EVIDENCE FOR A TWO-SITE BINDING MODEL^*

Guanghua Gao, Kirk C. Prutzman, Michelle L. King, Danielle M. Scheswohl, Eugene F. DeRose¶, Robert E. London¶, Michael D. Schaller||**, and Sharon L. Campbell||¶¶

From the Department of Biochemistry and Biophysics, the Department of Cell and Developmental Biology, ^||Lineberger Comprehensive Cancer Center, ^**Comprehensive Center for Inflammatory Disorders, and Carolina Cardiovascular Biology Center, University of North Carolina, Chapel Hill, North Carolina 27599 and ^¶Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is regulated by integrins. Upon activation, FAK generates signals that modulate crucial cell functions, including cell proliferation, migration, and survival. The C-terminal focal adhesion targeting (FAT) sequence mediates localization of FAK to discrete regions in the cell called focal adhesions. Several binding partners for the FAT domain of FAK have been identified, including paxillin. We have determined the solution structure of the avian FAT domain in complex with a peptide mimicking the LD2 motif of paxillin by NMR spectroscopy. The FAT domain retains a similar fold to that found in the unliganded form when complexed to the paxillin-derived LD2 peptide, an antiparallel four-helix bundle. However, noticeable conformational changes were observed upon the LD2 peptide binding, especially the position of helix 4. Multiple lines of evidence, including the results obtained from isothermal titration calorimetry, intermolecular nuclear Overhauser effects, mutagenesis, and protection from paramagnetic line broadening, support the existence of two distinct paxillin-binding sites on the opposite faces of the FAT domain. The structure of the FAT domain-LD2 complex was modeled using the program HADDOCK based on our solution structure of the LD2-bound FAT domain and mutagenesis data. Our model of the FAT domain-LD2 complex provides insight into the molecular basis of FAK-paxillin binding interactions, which will aid in understanding the role of paxillin in FAK targeting and signaling.

Received for publication, September 4, 2003 , and in revised form, November 18, 2003.

The atomic coordinates and structure factors (code 1qvx) 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 Grants CA90901 (to M. D. S.), DE13079 (to M. D. S.), and HL45100 (to M. D. S. and S. L. C.).

The on-line version of this article (available at http://www.jbc.org) contains Figs. 1 and 2.

To whom correspondence may be addressed: Dept. of Cell and Developmental Biology, CB 7090, University of North Carolina, Chapel Hill, NC 27599. Tel.: 919-966-0391; Fax: 919-966-1856; E-mail: crispy4{at}med.unc.edu.

^¶¶ To whom correspondence may be addressed: Dept. of Biochemistry and Biophysics, CB 7260, University of North Carolina, Chapel Hill, NC 27599. Tel.: 919-966-7139; Fax: 919-966-2852; E-mail: campbesl{at}med.unc.edu.

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