Oda M, Furukawa K, Ogata K, Sarai A, Ishii S, Nishimura Y, Nakamura H
Biomolecular Engineering Research Institute, Suita, Osaka, Japan.
Protein Eng. 1997 Dec;10(12):1407-14. doi: 10.1093/protein/10.12.1407.
The individual repeats, R2 and R3, of the minimum specific DNA-binding domain (R2R3) of c-Myb have very similar structures, with a helix-turn-helix variation motif, although their sequence identity in the tandem repeats is only 31%. From previous mutational and structural studies, the third helices in both repeats were shown to directly recognize the specific base sequence, PyAACG/TG. In order to elucidate the reason for the imperfection of the tandem repeats at amino acid positions other than the recognition helices, a series of R2R3 mutants was generated by swapping the helices and the N-terminus in R2 to those in R3. Consequently, the sequence composing the first helix of R2 was found to be essential for specific DNA-binding, in addition to the third recognition helix of R2. Further mutational studies revealed that the only indispensable residues in the first helix are Val103 and Val1O7, which are involved in the hydrophobic core of R2. These residues do not directly interact with the DNA, but they contribute to the correct formation of helix 1 and the characteristic packing of R2, which is slightly different from that of R3, and are required for specific base recognition through strong cooperativity with R3.
c-Myb最小特异性DNA结合结构域（R2R3）中的R2和R3这两个重复序列具有非常相似的结构，带有一个螺旋-转角-螺旋变异基序，尽管它们在串联重复序列中的序列同一性仅为31%。根据先前的突变和结构研究，两个重复序列中的第三个螺旋都被证明可直接识别特定碱基序列PyAACG/TG。为了阐明串联重复序列在识别螺旋以外的氨基酸位置存在缺陷的原因，通过将R2中的螺旋和N端与R3中的螺旋和N端进行交换，构建了一系列R2R3突变体。结果发现，除了R2的第三个识别螺旋外，构成R2第一个螺旋的序列对于特异性DNA结合也至关重要。进一步的突变研究表明，第一个螺旋中唯一不可或缺的残基是Val103和Val107，它们参与R2的疏水核心。这些残基并不直接与DNA相互作用，但它们有助于螺旋1的正确形成以及R2的特征性堆积，R2的堆积与R3略有不同，并且通过与R3的强协同作用实现特异性碱基识别是必需的。
