Garske Adam L, Craciun Gheorghe, Denu John M
Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706-1532, USA.
Biochemistry. 2008 Aug 5;47(31):8094-102. doi: 10.1021/bi800766k. Epub 2008 Jul 11.
Histone modifications modulate chromatin structure and function. A posttranslational modification-randomized, combinatorial library based on the first 21 residues of histone H4 was designed for systematic examination of proteins that interpret a histone code. The 800-member library represented all permutations of most known modifications within the N-terminal tail of histone H4. To determine its utility in a protein binding assay, the on-bead library was screened with an antibody directed against phosphoserine 1 of H4. Among the hits, 59 of 60 sequences were phosphorylated at S1, while 30 of 30 of those selected from the nonhits were unphosphorylated. A 512-member version of the library was then used to determine the binding specificity of the double tudor domain of hJMJD2A, a histone demethylase involved in transcriptional repression. Global linear least-squares fitting of modifications from the identified peptides (40 hits and 34 nonhits) indicated that methylation of K20 was the primary determinant for binding, but that phosphorylation and acetylation of neighboring sites attenuated the interaction. To validate the on-bead screen, isothermal titration calorimetry was performed with 13 H4 peptides. Dissociation constants ranged from 1 mM to 1 microM and corroborated the screening results. The general approach should be useful for probing the specificity of any histone-binding protein.
组蛋白修饰可调节染色质的结构和功能。基于组蛋白H4的前21个残基设计了一个翻译后修饰随机组合文库，用于系统检测解读组蛋白密码的蛋白质。这个包含800个成员的文库代表了组蛋白H4 N端尾巴内大多数已知修饰的所有排列组合。为了确定其在蛋白质结合测定中的效用，用针对H4第1位磷酸化丝氨酸的抗体对珠上文库进行筛选。在筛选出的序列中，60个序列中有59个在S1位点发生了磷酸化，而从未筛选出的序列中选取的30个序列均未发生磷酸化。然后使用该文库的一个包含512个成员的版本来确定参与转录抑制的组蛋白去甲基化酶hJMJD2A的双 Tudor 结构域的结合特异性。对鉴定出的肽段（40个筛选出的序列和34个未筛选出的序列）的修饰进行全局线性最小二乘法拟合表明，K20的甲基化是结合的主要决定因素，但相邻位点的磷酸化和乙酰化会减弱这种相互作用。为了验证珠上筛选结果，对13个H4肽段进行了等温滴定量热法测定。解离常数范围从1 mM到1 microM，证实了筛选结果。这种通用方法对于探究任何组蛋白结合蛋白的特异性应该是有用的。
