Department of Chemistry, Levine Science Research Center, Duke University, Durham, NC 27708, USA.
Biochemistry. 2011 Feb 1;50(4):546-57. doi: 10.1021/bi101776t. Epub 2010 Dec 30.
Flavin-dependent histone demethylases catalyze the posttranslational oxidative demethylation of mono- and dimethylated lysine residues, producing formaldehyde and hydrogen peroxide in addition to the corresponding demethylated protein. In vivo, histone demethylase LSD1 (KDM1; BCH110) is a component of the multiprotein complex that includes histone deacetylases (HDAC 1 and 2) and the scaffolding protein CoREST. Although little is known about the affinities of or the structural basis for the interaction between CoREST and HDACs, the structure of CoREST(286-482) bound to an α-helical coiled-coil tower domain within LSD1 has recently been reported. Given the significance of CoREST in directing demethylation to specific nucleosomal substrates, insight into the molecular basis of the interaction between CoREST and LSD1 may suggest a new means of inhibiting LSD1 activity by misdirecting the enzyme away from nucleosomal substrates. Toward this end, isothermal titration calorimetry studies were conducted to determine the affinity and thermodynamic parameters characterizing the binding interaction between LSD1 and CoREST(286-482). The proteins tightly interact in a 1:1 stoichiometry with a dissociation constant (K(d)) of 15.9 ± 2.07 nM, and their binding interaction is characterized by a favorable enthalpic contribution near room temperature with a smaller entropic penalty at pH 7.4. Additionally, one proton is transferred from the buffer to the heterodimeric complex at pH 7.4. From the temperature dependence of the enthalpy change of interaction, a constant-pressure heat capacity change (ΔC(p)) of the interaction was determined to be -0.80 ± 0.01 kcal mol(-1) K(-1). Notably, structure-driven truncation of CoREST revealed that the central binding determinant lies within the segment of residues 293-380, also known as the CoREST "linker" region, which is a central isolated helix that interacts with the LSD1 coiled-coil tower domain to create a triple-helical bundle. Thermodynamic parameters obtained from the binding between LSD1 and the linker region of CoREST are similar to those obtained from the interaction between LSD1 and CoREST(286-482). These results provide a framework for understanding the molecular basis of protein-protein interactions that govern nucleosomal demethylation.
黄素依赖的组蛋白去甲基酶催化单甲基化和二甲基化赖氨酸残基的翻译后氧化去甲基化，除了相应的去甲基化蛋白外，还产生甲醛和过氧化氢。在体内，组蛋白去甲基酶 LSD1（KDM1；BCH110）是包含组蛋白去乙酰化酶（HDAC1 和 2）和支架蛋白 CoREST 的多蛋白复合物的组成部分。尽管人们对 CoREST 与 HDACs 之间的相互作用的亲和力或结构基础知之甚少，但最近报道了 CoREST（286-482）与 LSD1 内的α-螺旋卷曲螺旋塔结构域结合的结构。鉴于 CoREST 在将去甲基化引导至特定核小体底物中的重要性，深入了解 CoREST 与 LSD1 之间的相互作用的分子基础可能会提出一种通过将酶从核小体底物上误导来抑制 LSD1 活性的新方法。为此，进行了等温热滴定法研究，以确定 LSD1 与 CoREST（286-482）之间结合相互作用的亲和力和热力学参数。这些蛋白质以 1:1 的化学计量紧密相互作用，解离常数（Kd）为 15.9±2.07 nM，并且它们的结合相互作用在室温附近表现出有利的焓贡献，在 pH7.4 时具有较小的熵罚。此外，在 pH7.4 时，从缓冲液中向异源二聚体复合物转移一个质子。从相互作用的焓变的温度依赖性确定相互作用的等压热容变化（ΔCp）为-0.80±0.01 kcal mol-1 K-1。值得注意的是，结构驱动的 CoREST 截断揭示了中央结合决定因素位于残基 293-380 之间的片段内，也称为 CoREST“连接”区域，这是一个与 LSD1 卷曲螺旋塔结构域相互作用的中央孤立螺旋，形成一个三螺旋束。从 LSD1 与 CoREST 的连接区域之间的结合获得的热力学参数与从 LSD1 与 CoREST（286-482）之间的相互作用获得的热力学参数相似。这些结果为理解控制核小体去甲基化的蛋白质-蛋白质相互作用的分子基础提供了一个框架。
