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Structural basis of tandemly connected engrailed homeodomains for designing a DNA binding protein array.
https://repo.qst.go.jp/records/77184
https://repo.qst.go.jp/records/771846e5e79cf-a56c-4bfa-8ec0-39772cdd1489
| Item type | 会議発表用資料 / Presentation(1) | |||||
|---|---|---|---|---|---|---|
| 公開日 | 2019-10-01 | |||||
| タイトル | ||||||
| タイトル | Structural basis of tandemly connected engrailed homeodomains for designing a DNA binding protein array. | |||||
| 言語 | ||||||
| 言語 | eng | |||||
| 資源タイプ | ||||||
| 資源タイプ識別子 | http://purl.org/coar/resource_type/c_c94f | |||||
| 資源タイプ | conference object | |||||
| アクセス権 | ||||||
| アクセス権 | metadata only access | |||||
| アクセス権URI | http://purl.org/coar/access_right/c_14cb | |||||
| 著者 |
Sunami, Tomoko
× Sunami, Tomoko× Hirano, Yuu× Tamada, Taro× Kono, Hidetoshi× Sunami, Tomoko× Hirano, Yuu× Tamada, Taro× Kono, Hidetoshi |
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| 抄録 | ||||||
| 内容記述タイプ | Abstract | |||||
| 内容記述 | Genome editing has become important tools not only for basic biological research but also clinical appreciations. Currently used systems such as TALEN and CRISPR/Cas9 have a potential limitation for further applications, which is caused by their large molecular weight. Therefore, a novel molecular scaffold to overcome their limitation has been desired. For this purpose, we selected the engrailed homeodomain (EHD), because the domain composed of only ∼60 amino acids can recognize 6 base pairs. In our previous work1, we have evaluated whether two domains connected with a several length of linkers (EHD2) can recognize the tandem target sites in E.coli cells, in order to clarify that EHD2 can work as an array, as other genome editing enzymes. The key finding was that the tandem target site was recognized by EHD2 when the conserved arginine 53 of the EHD was mutated to alanine in the tandem proteins ((R53A)2), while the tandem proteins without the mutation mainly bound to the monomeric site. To reveal the molecular mechanisms for recognition of the tandem target site, we determined a crystal structure of (R53A)2-DNA complex. We obtained 1.6 Å crystal structure of (R53A)2 bound to a 12 mer DNA, after trying co-crystallization of (R53A)2 with 12 to 22 bps of DNA. The individual EHDs adopt the typical homeodomain fold. One domain has base specific interactions with a cognate DNA, however, the other domain has weak interactions with the phosphates of DNA to work for crystal packing. A close-up view of the mutation site in the domain bound to cognate DNA and that of previously solved monomeric engrailed homeodomain2 (wt) are shown in Figure 1. We created R53A mutant to reduce binding affinity by losing the hydrogen bonds with the phosphate which is observed in the wild type structure. In the wild type EHD, base-specific interactions of I47, K50 and N51 with DNA in the major groove are important for affinity and specificity to DNA. We found that these interactions were completely preserved in (R53A)2. Together with the biochemical data, we conclude that (R53A)2 realizes the precise recognition of the tandem target site in cell by concurrent binding of the two individual EHD domains because the binding of the two domains properly compensates the decrease in affinity of the individual R53A EHD to the monomeric target DNA, but does not too much. |
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| 会議概要（会議名, 開催地, 会期, 主催者等） | ||||||
| 内容記述タイプ | Other | |||||
| 内容記述 | International Symposium on Diffraction Structure Biology 2019 | |||||
| 発表年月日 | ||||||
| 日付 | 2019-10-18 | |||||
| 日付タイプ | Issued | |||||
