Regulation of Ets-1 DNA binding

Autoinhibitory domains are increasingly being recognized as important means for proteins to regulate their activity in cis based on cellular cues. The transcription factor Ets-1 is regulated by an autoinhibitory module that represses the DNA binding of the ETS domain by hindering the structural rearrangement that accompanies DNA binding. However, the autoinhibitory module does not simply inhibiting binding; it serves as a versatile integrator of cell signals, including Ca2+-induced phosphorylation. This thesis explores the mechanism of autoinhibition of DNA binding in Ets-1, particularly in response to phosphorylation. Initial structural studies define the interface between the autoinhibitory module and the ETS domain. A purified multiply phosphorylated Ets-1 fragment, ?N2445P, is used to demonstrate that phosphorylation induced stabilization of the ETS domain and inhibitory module are correlated with a reduction in DNA-binding affinity. NMR spectrometry experiments reveal a dynamic hydrophobic network that connects the inhibitory module to the DNA-binding interface to form a concerted, regulatable unit. Together, these data support an allosteric model of Ets-1 DNA binding in which a dynamic hydrophobic network translates the level of phosphorylation into DNA-binding affinity by modulating the equilibrium between active and inactive conformations. Mutational analysis of the phosphorylated serine rich region indicates that multiple phosphates contribute additively to the inhibition, allowing variable control of DNA binding depending on phosphorylation state. Surprisingly, the serine rich region, which serves as an allosteric effector, is unstructured and highly mobile. Thus, rather than forming a simple on/off switch, this allosteric mechanism allows adjustable control of activity by combining flexible and folded modules. These results suggest that conformation coupling to transient interactions may provide a general mechanism of action of flexible regulatory segments.