Inhibitory module of Ets-1 allosterically regulates DNA binding through a dipole-facilitated phosphate contact.

DNA binding of the transcription factor Ets-1 is negatively regulated by three inhibitory helices that lie near the ETS domain. The current model suggests that this negative regulation, termed autoinhibition, is caused by the energetic expense of a DNA-induced structural transition that includes the unfolding of one inhibitory helix. This report investigates the role of helix H1 of the ETS domain in the autoinhibition mechanism. Previous structural studies modeled the inhibitory helices packing together and connecting with helix H1, suggesting a role of this helix in the configuration of an inhibitory module. Recently, high-resolution structures of the ETS domain-DNA interface indicate that the N terminus of helix H1 directly contacts DNA. The contact, which is augmented by the macrodipole of helix H1, consists of a hydrogen bond between the amide NH of leucine 337 in helix H1 and the oxygen of a corresponding phosphate. We propose that this hydrogen bond positions helix H1 to be a link between autoinhibition and DNA binding. Four independent approaches tested this hypothesis. First, the hydrogen bond was disrupted by removal of the phosphate in a missing phosphate analysis. Second, base pairs that surround the helix H1-contacting phosphate and appear to dictate DNA backbone conformation were mutated. Next, a hydrophobic residue in helix H1 that is expected to position the N terminus of the helix was altered. Finally, a residue on the surface of helix H1 that may contact the inhibitory elements was changed. In each case DNA binding and autoinhibition was affected. Taken together, the results demonstrate the role of the dipole-facilitated phosphate contact in DNA binding. Furthermore, the findings support a model in which helix H1 links the inhibitory elements to the ETS domain. We speculate that this helix, which is conserved in all Ets proteins, provides a common route to regulation.