Protein Science zyxwvutsrqpon (1996), 5:296-309. Cambridge University Press. Printed in the USA. Copyright zyxwvutsrqp 0 1996 The Protein Society Structural coupling of the inhibitory regions flanking the ETS domain of murine Ets-1 zyxw JACK J. SKALICKY,1*3 LOGAN W. DONALDSON,' JEANNINE M. PETERSEN,' BARBARA J. GRAVES,' AND LAWRENCE P. McINTOSH' zyxwvu ' The Department of Biochemistry and Molecular Biology and the Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 123, Canada The Department of Oncological Sciences, Division of Molecular Biology and Genetics, University of Utah School of Medicine, Salt Lake City, Utah 84132 (RECEIVED October 9, 1995; ACCEPTED November 11, 1995) Abstract Several members of the zyxwvutsrq ets gene family of transcription factors show negative regulation of DNA binding by in- tramolecular interactions. A structural mechanism for this auto-inhibition is investigated using a 161-residue N-terminal deletion mutant of murine Ets-1, Ets-1AN280. This protein shows a similar reduced affinity for DNA as native Ets-1 because it contains the ETS domain in context of the flanking amino- and carboxy-terminal re- gions that together mediate repression of DNA binding. The secondary structure of Ets-IAN280 was determined using NMRchemical shift, NOE, J coupling, and amide hydrogen exchange information. In addition to the winged helix-turn-helix ETS domain, Ets-1AN280 contains two a-helices in the amino-terminalinhibitory region and one a-helix in the carboxy-terminal inhibitory region. Chemical shift comparisons were made between this protein and an activated form of Ets-1 lacking the amino-terminal inhibitory region. The spectral differences demonstrate that the amino- and carboxy-terminal inhibitory sequences are structurally coupled to one another, thus explaining the observation that bothregions are required for the repression of DNA binding. Furthermore, these data show that the inhibitory sequences also interact directly with the first helix of the intervening ETS domain, thereby pro- viding a pathway for the repression of DNA binding. These results lead to a model of an inhibitory module in Ets-1 composed of both the amino- and carboxy-terminal regions interfaced with the ETS domain. This estab- lishes the structural framework for understanding the intramolecular inhibition of Ets-1 DNA binding. Keywords: allosteric; DNA binding; ETS domain; Ets-1 and Ets-2; intramolecular inhibition; NMR; protein struc- ture; winged helix-turn-helix DNA binding, activation, and oligomerization associated with a particular transcription factor may be governed by its subcel- lular localization, posttranslational modifications, ligand bind- ing,andprotein-proteininteractions. Recently, data have Reprint requests to: Lawrence McIntosh, Department of Biochem- istry, 2146 Health Sciences Mall, University of British Columbia, Van- couver, British Columbia V6T 123, Canada;e-mail: mcintosh@otter. biochem.ubc.ca. Present address: Department of Chemistry, SUNY-Buffalo, Buf- falo, New York 14260-3000. Abbreviations: ets, E26 transformation specific; Ets-1AC428, the carboxy-terminal deletion fragment of murine Ets-1 consisting of resi- dues 1-428; Ets-1AN280, the amino-terminal deletion fragment of murine Ets-1 consisting of residues 280-440; Ets-1AN331, the amino- terminal deletion fragment of murine Ets-1 consisting of residues 33 1- 440; FID, free induction decay; HSQC, heteronuclear single quantum correlation; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy; IPTG, zyxwvutsr isopropyl-8-D-thiogalactopyrano- side; DTT, dithiothreitol; pH*, the observed pH meter reading without correction for isotope effects; wHTH, winged helix-turn-helix; HSF, heat shock transcriptionfactors; DSS, 2,2-dimethyl-2-silapentane-5-sul- fonate, sodium salt. emerged demonstrating that transcription factors can also neg- atively autoregulate their own activity. This phenomenon of in- tramolecular inhibition is supported by a growing body of evidence suggesting that the functional characteristics of an iso- lated transcription factor domain can be different when com- pared with those of the intact protein. For example, deletions or mutations outside the DNA-binding domains of p53, NFKB, and TATA-Binding Protein (TBP) substantially alter the affin- ity of these regulatory proteins for DNA (Lieberman et al., 1991; Hupp et al., 1992; Grimm & Baeuerle, 1993). In addition, tri- merization of human and Drosophila heat shock transcription factors, HSF, is suppressed by intramolecular coiled-coil inter- actions (Rabindran et al., 1993). Repression of the activation domain of the transcription factor CRP2 (CIEBPP) has also been shown to be modulated by adjacent sequences (Kowenz- Leutz et al., 1994; Williams et al., 1995). These observations challenge the view of transcription factors as composite proteins comprised of autonomous functional domains. Furthermore, it stresses the importance of characterizing the domains from these proteins both in isolation and in their native structural context 296