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J. Biol. Chem., Vol. 280, Issue 8, 7088-7099, February 25, 2005

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The Structural and Dynamic Basis of Ets-1 DNA Binding Autoinhibition^*

Gregory M. Lee, Logan W. Donaldson¶, Miles A. Pufall||, Hyun-Seo Kang, Isabelle Pot, Barbara J. Graves||, and Lawrence P. McIntosh, A CIHR Scientist**

From the Department of Biochemistry and Molecular Biology, Department of Chemistry, and The Biotechnology Laboratory, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada and the ^||Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84112

The transcription factor Ets-1 is regulated by the allosteric coupling of DNA binding with the unfolding of an -helix (HI-1) within an autoinhibitory module. To understand the structural and dynamic basis for this autoinhibition, we have used NMR spectroscopy to characterize Ets-1N301, a partially inhibited fragment of Ets-1. The NMR-derived Ets-1N301 structure reveals that the autoinhibitory module is formed predominantly by the hydrophobic packing of helices from the N-terminal (HI-1, HI-2) and C-terminal (H4, H5) inhibitory sequences, along with H1 of the intervening DNA binding ETS domain. The intramolecular interactions made by HI-1 in Ets-1N301 are similar to the intermolecular contacts observed in the crystal structure of an Ets-1N300 dimer, confirming that the latter represents a domain-swapped species. ¹⁵N relaxation studies demonstrate that the backbone of the N-terminal inhibitory sequence is mobile on the nanosecond-picosecond and millisecond-microsecond time scales. Furthermore, hydrogen exchange measurements reveal that amide protons in helices HI-1 and HI-2 exchange with water at rates only 15- and 75-fold slower, respectively, than predicted for an unfolded polypeptide. These findings indicate that inhibitory helices are only marginally stable even in the absence of DNA. The energetic coupling of DNA binding with the facile unfolding of the labile HI-1 provides a mechanism for modulating Ets-1 DNA binding activity via protein partnerships, post-translational modifications, or mutations. Ets-1 autoinhibition illustrates how conformational equilibria within structural domains can regulate macromolecular interactions.

Received for publication, September 17, 2004 , and in revised form, December 3, 2004.

The atomic coordinates of the Ets-1N301 ensemble (code 1R36) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswich, NJ (http://www.rcsb.org/).

The NMR chemical shift list (accession code 5991) has been deposited in the BioMagResBank at www.bmrb.wisc.edu/.

^* This work was supported by grants from the National Cancer Institute of Canada with funds from the Canadian Cancer Society (to L. P. M.), the National Institutes of Health Grants GM38663 (to B. J. G.) and CA24014 (to the Huntsman Cancer Institute), and CA93247 and GM08537 for training grant support (to M. A. P.). 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 Materials.

Both authors contributed equally to this work.

^¶ Present address: Dept. of Biology, York University, Toronto, ON M3J 1P3, Canada.

^** To whom correspondence should be addressed. Tel.: 604-822-3341; Fax: 604-822-5227; E-mail: mcintosh{at}otter.biochem.ubc.ca.

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