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首页> 外文期刊>Acta crystallographica. Section D, Structural biology >Structural basis for designing an array of engrailed homeodomains
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Structural basis for designing an array of engrailed homeodomains

机译：结构基础设计的数组十字homeodomains

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Small DNA-binding proteins that target desired sequences have the potential to act as a scaffold for molecular tools such as genome editing. In this study, an engrailed homeodomain (EHD) was chosen and it was evaluated whether it could be used as a molecular module that can connect to itself to recognize a longer target sequence. It was previously shown that two EHDs connected by a linker (EHD_2) recognize a target sequence twice as long as that recognized by a single EHD in cells only when Arg53 in each EHD in the tandem protein is mutated to alanine {(EHD[R53A])_2}. To investigate the recognition mechanism of (EHD[R53A])_2, the crystal structure of the (EHD[R53A])_2-DNA complex was determined at 1.6 A resolution. The individual EHDs were found to adopt the typical homeodomain fold. Most importantly, the base-specific interactions in the major groove necessary for the affinity/specificity of wild-type EHD were preserved in (EHD[R53A])_2. Bacterial assays confirmed that the base-specific interactions are retained under cellular conditions. These observations indicate that the R53 A mutation only causes a loss of the arginine-phosphate interaction at the protein-DNA interface, which reduces the DNA-binding affinity compared with the wild type. It is therefore concluded that (EHD[R53A])2 precisely recognizes tandem target sites within cells, enabling the individual EHDs to concurrently bind to the target sites with modest binding affinity. This suggests that modulation of the binding activity of each EHD is vital to construct a protein array that can precisely recognize a sequence with multiple target sites.
机译：小的dna结合蛋白质所需的目标序列有可能作为支架分子工具,如基因组编辑。这项研究中,一个十字的homeodomain (EHD)选择和评估是否可以作为一个分子模块,它可以连接到自己认识到更长的目标序列。以前显示两个EHDs连接的吗链接器(EHD_2)识别目标序列的两倍由一个EHD只要承认细胞只有当每个EHD Arg53串联蛋白质是突变为丙氨酸{(EHD [R53A]) _2}。调查的识别机制(EHD [R53A]) _2的晶体结构(EHD [R53A]) _2-DNA复杂决心在1.6决议。采用典型的homeodomain褶皱。重要的是,base-specific交互主要槽所必需的亲和力/野生型EHD的特异性保存在(EHD (R53A) _2)。确认base-specific交互保留在细胞的条件下。观测表明,R53突变只有arginine-phosphate造成损失在protein-DNA接口交互,这减少了与dna结合亲和力野生型。(EHD [R53A]) 2精确识别串联的目标网站的细胞内,使个人EHDs并发地绑定到目标站点适度的亲和力。调制的绑定每个EHD的活动构造一个蛋白质阵列可以至关重要准确地识别一个与多个序列目标网站。

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