Summary Binding of the transcription factor Ets-1 to DNA is allosterically regulated by a serine rich region (SRR) that modulates the dynamic character of the adjacent structured DNA-binding ETS domain and its flanking autoinhibitory elements. Multi-site phosphorylation of the flexible SRR in response to Ca+2 signaling mediates variable regulation of Ets-1 DNA-binding affinity. In this study, we further investigated the mechanism of this regulation. First, thermal and urea denaturation experiments demonstrated that phosphorylation of the predominantly unstructured SRR imparts enhanced thermodynamic stability on the well-folded ETS domain and its inhibitory module. We next identified a minimal fragment (residues 279–440) that exhibits both enhanced autoinhibition of Ets-1 DNA-binding and allosteric reinforcement by phosphorylation. To test for intramolecular interactions between the SRR and the rest of the fragment that were not detectable by 1H-1H NOE measurements, paramagnetic relaxation enhancements were performed using a Cu2+ bound to the N-terminal ATCUN motif. Increased relaxation detected for specific amides and methyls revealed a preferential interaction surface for the flexible SRR extending from the inhibitory module to the DNA binding interface. Phosphorylation enhanced the localization of the SRR to this surface. We therefore hypothesize that the positioning of the SRR at the DNA binding interface and its role in shifting Ets-1 to an inhibited conformation are linked. In particular, transient interactions dampen the conformational flexibility of the ETS domain and inhibitory module required for high affinity binding, as well as possibly occlude the DNA interaction site. Surprisingly, the phosphorylation-dependent effects were relatively insensitive to changes in ionic strength, suggesting that electrostatic forces are not the dominant mechanism for mediating these interactions. The results of this study highlight the role of flexibility and transient binding in the variable regulation of Ets-1 activity.

The E26 transformation-specific (Ets-1) transcription factor is autoinhibited by a conformationally disordered serine-rich region (SRR) that transiently interacts with its DNA-binding ETS domain. In response to calcium signaling, autoinhibition is reinforced by calmodulin-dependent kinase II phosphorylation of serines within the SRR. Using mutagenesis and quantitative DNA-binding measurements, we demonstrate that phosphorylation-enhanced autoinhibition requires the presence of phenylalanine or tyrosine (ϕ) residues adjacent to the SRR phosphoacceptor serines. The introduction of additional phosphorylated Ser-ϕ-Asp, but not Ser-Ala-Asp, repeats within the SRR dramatically reinforces autoinhibition. NMR spectroscopic studies of phosphorylated and mutated SRR variants, both within their native context and as separate trans-acting peptides, confirmed that the aromatic residues and phosphoserines contribute to the formation of a dynamic complex with the ETS domain. Complementary NMR studies also identified the SRR-interacting surface of the ETS domain, which encompasses its positively charged DNArecognition interface and an adjacent region of neutral polar and nonpolar residues. Collectively, these studies highlight the role of aromatic residues and their synergy with phosphoserines in an intrinsically disordered regulatory sequence that integrates cellular signaling and gene expression.transcription factor regulation | protein dynamics | intrinsically disordered region | fuzz complex I ntrinsically disordered protein regions (IDRs) are increasingly recognized for their prevalence in the eukaryotic proteome and their roles in normal biological processes, as well as in disease (1-3). IDRs serve as flexible linker sequences between modular domains and as key components of complex protein-interaction networks. These sequences are often sites of posttranslational modifications, and their plasticity enables accessible and reversible interactions necessary for the integration of cellular signals.The autoinhibition of the E26 transformation-specific (Ets-1) transcription factor provides an illuminating example of how a flexible, disordered region can modulate the regulatable DNAbinding ETS domain and thereby tune it for biological control by calcium-dependent phosphorylation and cooperative protein partnerships (4). The inhibitory module (IM) of Ets-1 is composed of four α-helices (HI-1, HI-2, H4, and H5) that pack onto the ETS domain distal from the DNA interface (Fig. 1A) (5). Helix HI-1 is marginally stable and unfolds on DNA binding, thus implicating an allosteric mechanism of inhibition (6, 7). The modest twofold repression afforded by the IM is increased to ∼20-fold by an intrinsically disordered serine-rich region (SRR) (8, 9). The dynamic SRR interacts transiently with both the ETS domain DNA-binding interface and the IM, and thus plays steric and allosteric inhibitory roles (10). Phosphorylation of five SRR serines by calmodulindependent kinase II (CaMKII) leads to a dramatic ∼500-fold autoinhibition (11). In paral...