Structural basis of DNA binding and autoinhibition for the eukaryotic transcription factor ETS-1

The ets family of transcription factors represents a paradox in the study of DNA binding proteins. How can regulatory proteins that share the same DNA binding properties exert the distinguishable effects necessary to execute gene regulation? As a framework for addressing this question, the molecular basis of DNA binding for Ets-1, the founding member of the ets family, was determined. Two N-terminal deletion polypeptides of Ets-1 that retained either DNA binding (DeltaN331) or DNA binding and autoinhibition (DeltaN280) were utilized for these studies. Determination of the nuclear magnetic resonance (NMR) secondary structure for the Ets-1 DNA binding domain (ETS domain) classified the ets proteins into a structural family of DNA binding proteins termed the winged helix-turn-helix (wHTH) superfamily. Tertiary structure analysis of the Ets-1 ETS domain confirmed the wHTH DNA binding motif and showed that the ETS domain is composed of a three-helix bundle packed against a four-stranded antiparallel beta-sheet. A helix-turn-helix DNA binding motif is found within the three-helix bundle. Insight into the mode of DNA binding utilized by Ets-1 and other ets proteins was obtained by comparison to previously described cocrystal structures for wHTH proteins. Additional insights into ets protein DNA binding specificity come from structural studies of Ets-1 autoinhibition. Quantitative DNA binding assays defined a minimal region required for Ets-1 DNA binding inhibition. Thermal denaturation studies demonstrated that the functions of inhibition and DNA binding are coupled within a single structural domain. NMR secondary structure analysis confirmed this finding and showed that the Ets-1 inhibitory domain is composed of four alpha helices; two alpha helices in the N-terminal inhibitory region, one alpha helix in the C-terminal inhibitory region, and helix one in the ETS domain. Furthermore, these studies demonstrated that the alpha helices within the N- and C-terminal inhibitory regions of Ets-1 are structurally coupled to each other and to the ETS domain. Upon DNA binding, a unique conformational change involving unfolding of an alpha helix within the N-terminal inhibitory region was observed. This provided evidence that disruption of the inhibitory module occurs on DNA binding. A model is presented whereby interactions with a protein partner could serve to stabilize the de-repressed Ets-1 conformation. Thus, specificity of binding for members of the ets family that display autoinhibition of DNA binding could be determined via interaction with a protein partner. These findings provide important insight into the molecular basis of cooperative partnerships between DNA binding proteins and the conformational changes that regulate transcription factor function.