STRUCTURAL COUPLING OF THE INHIBITORY REGIONS FLANKING THE ETS DOMAINOF MURINE ETS-1

Several members of the ets gene family of transcription factors show n egative regulation of DNA binding by intramolecular interactions. A st ructural mechanism for this auto-inhibition is investigated using a 16 1-residue N-terminal deletion mutant of murine Ets-1, Ets-1 Delta N280 . 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 regions that together mediate repression of DNA binding. The secondary structure of Ets-1 Delta N280 was determined us ing NMR chemical shift, NOE, J coupling, and amide hydrogen exchange i nformation. In addition to the winged helix-turn-helix ETS domain, Ets -1 Delta N280 contains two alpha-helices in the amino-terminal inhibit ory region and one alpha-helix in the carboxy-terminal inhibitory regi on. Chemical shift comparisons were made between this protein and an a ctivated form of Ets-1 lacking the amino-terminal inhibitory region. T he spectral differences demonstrate that the amino- and carboxy-termin al inhibitory sequences are structurally coupled to one another, thus explaining the observation that both regions are required for the repr ession of DNA binding. Furthermore, these data show that the inhibitor y sequences also interact directly with the first helix of the interve ning ETS domain, thereby providing 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 establishes the structural framework for un derstanding the intramolecular inhibition of Ets-1 DNA binding.