The affinity of Ets-1 for DNA is modulated by phosphorylation through transient interactions of an unstructured region.
The affinity of Ets-1 for DNA is modulated by phosphorylation through transient interactions of an unstructured region.
复制标题
DOI：
10.1016/j.jmb.2008.07.064
复制
发表时间：
2008-10-17
影响因子：
5.6
通讯作者：
McIntosh LP
中科院分区：
文献类型：
--
作者：
Lee GM;Pufall MA;Meeker CA;Kang HS;Graves BJ;McIntosh LP
转录因子Ets-1与DNA的结合受富含丝氨酸的区域（SRR）的变构调节，所述富含丝氨酸的区域调节相邻结构化DNA结合ETS结构域及其侧翼的自身抑制元件的动态特性。柔性SRR响应Ca+2信号的多位点磷酸化介导Ets-1 DNA结合亲和力的可变调节。在本研究中，我们进一步研究了这种调节的机制。首先，热变性和尿素变性实验表明，磷酸化的主要是非结构化的SRR赋予增强的热力学稳定性的良好折叠的ETS域及其抑制模块。我们接下来鉴定了一个最小片段（残基279-440），其表现出Ets-1 DNA结合的增强的自抑制和通过磷酸化的变构增强。为了测试SRR与通过1H-1H NOE测量无法检测到的其余片段之间的分子内相互作用，使用与N-末端ATCUN基序结合的Cu 2+进行顺磁弛豫增强。增加松弛检测特定的酰胺和甲基揭示了一个优先的相互作用表面的柔性SRR从抑制模块延伸到DNA结合界面。磷酸化增强了SRR在该表面的定位。因此，我们假设，在DNA结合界面的SRR的定位和它的作用转移Ets-1的抑制构象连接。特别地，瞬时相互作用抑制ETS结构域和高亲和力结合所需的抑制模块的构象灵活性，以及可能封闭DNA相互作用位点。令人惊讶的是，磷酸化依赖的影响是相对不敏感的离子强度的变化，这表明静电力是不是介导这些相互作用的主导机制。本研究的结果突出了灵活性和瞬时结合在Ets-1活性的可变调节中的作用。
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.