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Structural studies of the ETS domain

University of British Columbia

This thesis describes the cloning, overexpression, biophysical and structural analyses of fragments from murine Ets-1 that comprise the ETS domain, a motif responsible for the sequence-specific DNA binding activity of the ets family. Multidimensional nuclear magnetic resonance (NMR) techniques feature prominently throughout this study. From the tertiary structure of a 110 residue Ets-1 fragment, the ETS domain is demonstrated to possess a winged helix-turn-helix fold. As the name suggests, the winged helix-turnhelix motif is comprised of a helix-turn-helix motif and a characteristic (3-sheet component which pack to form a compact domain. In some members of the winged helix-turn-helix family, a mobile wing-like (3-turn supplies base-specific contacts to augment contacts made by the recognition helix. The ETS domain of Ets-1 is flanked by amino- and carboxy-terminal sequences that cooperate to attenuate DNA binding affinity by approximately twenty fold. Through structural analysis of a larger, 142 residue fragment of Ets-1, the flanking inhibitory sequences are shown to form a module comprised of two N-terminally located cc-helices, one C-terminally located ct-helix and one cc-helix from the ETS domain. An allosteric mechanism of repression is proposed as the inhibition module is located on different face of the ETS domain relative to the recognition helix. Dynamic information obtained from ¹⁵N relaxation and fast amide hydrogen exchange experiments support this mechanism of Ets-1 intramolecular inhibition.

Thesis/Dissertation

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2009-02-18

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http://hdl.handle.net/2429/4778

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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