The Affinity of Ets-1 for DNA is Modulated by Phosphorylation Through Transient Interactions of an Unstructured Region

Abstract

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 Ca²⁺ 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 Cu²⁺ bound to the N-terminal ATCUN motif. Increased relaxation detected for specific amide and methyl groups 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.

Introduction

Regions of intrinsic disorder are being identified with increasing frequency as targets of protein regulation.1, 2, 3 Two general features of these regions have been described: first, they adopt an ordered conformation in order to perform their function; and second, they are often sites of post-translational modifications, which either induce or disrupt the active conformation. For example, the tail of stathmin, which is disordered in its apo form, adopts a stable β-hairpin when bound to tubulin, leading to the disruption of microtubule formation. Phosphorylation of this region abolishes the hairpin, thus disrupting binding and allowing microtubule formation to proceed.⁴ Conversely, phosphorylation of the intrinsically disordered KID domain of the DNA-binding transcription factor CREB enhances recognition by the KIX domain of co-activator p300. The phosphorylated KID domain undergoes a disorder-to-order transition upon interaction with the KIX domain, forming two α-helices that provide a stable interface for association with p300 and allowing activation of transcription.⁵ In both cases, the change in the physicochemical properties of the modified protein conferred by addition of phosphates has a role in either inducing or disrupting the active structure. In contrast, our recent studies of the transcription factor Ets-1 show that a predominantly unstructured, multiply phosphorylated region acts as an intramolecular allosteric regulator of the well-folded DNA-binding domain.⁶ The interplay between the unstructured and structured regions of Ets-1 is the focus of this investigation.

The binding of Ets-1 to DNA is mediated by the ETS domain, which contains three α-helices and a four-stranded β-sheet, and is modulated by flanking regions that fold into an inhibitory module composed of four α-helices (Fig. 1).7, 8 The inhibitory module packs along a surface of the ETS domain at helix H1, opposite the helix (H2)-turn-helix(H3) DNA recognition interface. DNA binding is accompanied by a structural rearrangement that disrupts the inhibitory module, as highlighted most dramatically by the unfolding of the marginally stable N-terminal inhibitory helix HI-1.8, 9, 10 Proper packing of the inhibitory module against the ETS domain is required for autoinhibition, as demonstrated by the higher affinity of variants containing mutations that disrupt the hydrophobic core of the module.¹¹ The presence of an adjacent disordered serine-rich region (residues 244–300, termed the SRR) accentuates repression of the ETS domain and inhibitory modules (residues 301–440, also termed ΔN301 or the “regulatable unit”) and recapitulates binding of the full-length protein.⁶ Multi-site phosphorylation of the SRR in response to Ca²⁺ signaling further attenuates DNA binding,¹² with each added phosphate causing progressively increased repression.⁶ Protein partnerships, both heterotypic and homotypic, can also counteract autoinhibition, thereby enhancing DNA binding.13, 14, 15 Thus, Ets-1 displays a 1000-fold range in DNA binding affinity, from a state completely activated by deletion or biological partners to a fully phosphorylated, repressed state.

The mechanism of autoinhibition and its reinforcement by phosphorylation was partially elucidated by the discovery of the dynamic nature of the ETS domain and its inhibitory elements, even in the absence of DNA.6, 8 The degree of this internal motion correlates with the DNA-binding affinity of various deletion fragments and phosphorylated forms of Ets-1. Specifically, the presence of the SRR and its phosphorylation reduces the millisecond to microsecond timescale motions of the inhibitory module and ETS domain, as well as imparting its overall stabilization, as shown by increased protection from amide hydrogen exchange (HX).⁶ Along with chemical shift perturbation mapping, these NMR-based observations indicated that Ets-1 exists in a conformational equilibrium between at least two states. The active state, which can bind DNA, exhibits conformational mobility within HI-1 and HI-2 of the inhibitory module and, importantly, the DNA recognition helix H3.⁶ This led to the model that the dynamic nature of Ets-1 reflects the sampling of conformations necessary to bind to DNA recognition sites with high affinity, and that a reduction in these dynamics shifts the equilibrium to a more rigid, low-affinity state.

The shift between dynamic-active and rigid-inactive states is reminiscent of the classical allosteric transition between the relaxed and tense forms of a protein.¹⁶ However, distinct from this model, the phosphorylation-dependent exchange between the two states of Ets-1 is not all-or-none. Progressive mutation of phosphoacceptor sites within the SRR yielded species with progressively decreasing inhibition, indicating that the affinity of Ets-1 for DNA can be fine-tuned in vitro⁶ and in vivo¹⁷ by multi-site phosphorylation. Importantly, each added phosphate also shifts the equilibrium toward the inhibited conformation, further bolstering the idea that decreasing the active state population is the mode for reducing DNA affinity. Surprisingly, the phosphorylated SRR directs this conformational transition despite being predominantly unstructured and flexible on the sub-nanosecond timescale.⁶

To further our understanding of the mechanism of Ets-1 autoinhibition and to determine how structured and unstructured protein segments interact, we investigated several unresolved features of the allosteric model. Does the SRR interact directly with the ETS domain and inhibitory helices to mediate its stabilizing effect? Does this change upon phosphorylation? What is the physicochemical nature of this interaction between an unstructured, flexible region and a dynamic structured region? In particular, are electrostatic forces involved due to the addition of negative charges upon phosphorylation? To address these questions, we demonstrate that SRR phosphorylation increases the global thermodynamic stability of an Ets-1 fragment. Using NMR spectroscopy, we also show that the SRR is predominantly unstructured both before and after phosphorylation. However, addition of phosphates to the SRR subtly dampens its fast timescale motions detected by ¹⁵N relaxation, as well as restricting the conformational dynamics of the regulatable unit, as shown by reduced amide HX. Importantly, paramagnetic relaxation enhancement (PRE) measurements revealed that the flexible SRR localizes to a surface that extends from the inhibitory module to the DNA-binding interface, and that this localization is more persistent upon phosphorylation. These observations suggest that transient intramolecular interactions can affect the dynamic nature of the ETS domain and its inhibitory elements, thus supporting an allosteric mechanism of autoinhibition. The location of the SRR near the DNA-binding surface might also augment autoinhibition through a steric mechanism.