Thermodynamic and structural insights into CSL-DNA complexes
Thermodynamic and structural insights into CSL-DNA complexes
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DOI：
10.1002/pro.280
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发表时间：
2010-01-01
期刊：
影响因子：
8
通讯作者：
Kovall, Rhett A.
中科院分区：
文献类型：
--
作者：
Friedmann, David R.;Kovall, Rhett A.
Notch途径是一种细胞间信号机制，在整个发育和成体生物体的细胞命运决定中发挥着重要作用。Notch受体与配体的胞外络合最终导致基因表达的变化，该基因表达受该途径的核效应因子CSL(C-启动子结合因子1(CBF-1)、无毛抑制因子(SU(H))、LIN-12和GLP-1(LAG-1))的调节。CSL是一种DNA结合蛋白，参与对Notch信号反应的基因转录的抑制和激活。一个Notch靶基因是HES-1的毛发和增强子(HES-1)，它由一个启动子元件调控，该启动子元件由两个以头对头排列的CSL结合位点组成。虽然以前的研究已经确定了CSL的体内和共识结合部位，并且已经确定了这些络合物的晶体结构，但到目前为止，对CSL-DNA结合的能量学的定量描述尚不清楚。在这里，我们提供了CSL与组成HES-1启动子元件的两个单独位点之间相互作用的热力学和结构分析。我们的综合研究分析了结合随温度、盐和pH的变化，发现CSL对两个HES-1结合位点的亲和力存在适度但明显的差异。同样，我们的结构结果表明，总体上CSL以类似的方式结合了这两个DNA位点；然而，CSL和DNA的构象都发生了微小的变化。综上所述，我们的结果为了解CSL如何与体内DNA位点相互作用提供了定量和生物物理基础。
The Notch pathway is an intercellular signaling mechanism that plays important roles in cell fates decisions throughout the developing and adult organism. Extracellular complexation of Notch receptors with ligands ultimately results in changes in gene expression, which is regulated by the nuclear effector of the pathway, CSL (C-promoter binding factor 1 (CBF-1), suppressor of hairless (Su(H)), lin-12 and glp-1 (Lag-1)). CSL is a DNA binding protein that is involved in both repression and activation of transcription from genes that are responsive to Notch signaling. One well-characterized Notch target gene is hairy and enhancer of split-1 (HES-1), which is regulated by a promoter element consisting of two CSL binding sites oriented in a head-to-head arrangement. Although previous studies have identified in vivo and consensus binding sites for CSL, and crystal structures of these complexes have been determined, to date, a quantitative description of the energetics that underlie CSL-DNA binding is unknown. Here, we provide a thermodynamic and structural analysis of the interaction between CSL and the two individual sites that comprise the HES-1 promoter element. Our comprehensive studies that analyze binding as a function of temperature, salt, and pH reveal moderate, but distinct, differences in the affinities of CSL for the two HES-1 binding sites. Similarly, our structural results indicate that overall CSL binds both DNA sites in a similar manner; however, minor changes are observed in both the conformation of CSL and DNA. Taken together, our results provide a quantitative and biophysical basis for understanding how CSL interacts with DNA sites in vivo.