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Structural basis for designing an array of engrailed homeodomains.
Acta Crystallographica Section D ( IF 2.2 ) Pub Date : 2020-09-02 , DOI: 10.1107/s2059798320009237
Tomoko Sunami 1 , Yu Hirano 2 , Taro Tamada 2 , Hidetoshi Kono 1
Affiliation  

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 (EHD2) 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 Å 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 R53A 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.

中文翻译:

设计一系列纠缠的同源域的结构基础。

靶向所需序列的小DNA结合蛋白有潜力充当分子工具如基因组编辑的支架。在这项研究中,选择了一个复杂的同源域(EHD),并评估了它是否可以用作可以与自身连接以识别更长的靶序列的分子模块。有人曾表明,通过接头(EHD连接的两个EHDs 2)的两倍,只要该识别识别靶序列通过在电池的单EHD只有当Arg53在串联蛋白质每EHD被突变成丙氨酸{(EHD [R53A ]] 2 }。为了研究的识别机构(EHD [R53A])2,所述的晶体结构(EHD [R53A])2– DNA复合物的分辨率为1.6Å。发现各个EHD采用典型的同源域折叠。最重要的是,(EHD [R53A])2中保留了野生型EHD亲和力/特异性所必需的主要沟中的碱基特异性相互作用。细菌分析证实,碱基特异性相互作用在细胞条件下得以保留。这些观察结果表明,R53A突变仅导致蛋白质-DNA界面上精氨酸-磷酸盐相互作用的丧失,与野生型相比,这降低了DNA结合亲和力。因此得出的结论是(EHD [R53A])2精确识别细胞内的串联靶位点,从而使单个EHD能够以适度的结合亲和力同时结合至靶位点。这表明调节每个EHD的结合活性对于构建可以精确识别具有多个靶位点的序列的蛋白质阵列至关重要。
更新日期:2020-09-02
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