@article{oai:repo.qst.go.jp:00080159,
 author = {Tomoko, Sunami, and Yuu, Hirano, and Taro, Tamada, and Hidetoshi, Kono, and Tomoko, Sunami and Yuu, Hirano and Taro, Tamada and Hidetoshi, Kono},
 issue = {9},
 journal = {Acta Crystallographica Section D},
 month = {Aug},
 note = {Small DNA binding proteins to target desired sequences have the potential to become a scaffold of molecular tools such as genome-editing. In our study, engrailed homeodomain (EHD) has been chosen and evaluated whether it can work as a molecular module connected with each other to recognize the longer target sequence. It was previously shown that two EHDs connected with a linker (EHD2) recognizes a target sequence twice as long as a single EHD in cells only when arginine 53 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 R53A mutation causes only a loss of the arginine-phosphate interaction at the protein-DNA interface, which reduces the DNA binding affinity compared with the wild-type. We therefore conclude 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 for constructing a protein array that can precisely recognize a sequence with multiple target sites.},
 pages = {824--833},
 title = {Structural basis for an array of engrailed homeodomains},
 volume = {76},
 year = {2020}
}