Sasaki M, Ogata K, Hatanaka H, Nishimura Y
Graduate School of Integrated Science, Yokohama City University, Seto, Kanazawa-ku, Yokohama 236-0027, Japan.
J Biochem. 2000 Jun;127(6):945-53. doi: 10.1093/oxfordjournals.jbchem.a022710.
The DNA-binding domain of c-Myb consists of three imperfect tandem repeats, R1, R2, and R3. Each repeat contains three helices. The minimal DNA-binding domain is an R2R3 fragment. Here, we have examined the backbone dynamics of R2R3 in its DNA-bound form by NMR. Upon binding to DNA, the N- and C-termini, and the linker between R2 and R3 become less flexible. In the free form the third helix of R2 exhibits slow conformational exchange fluctuations owing to a cavity in the hydrophobic core of R2. Upon binding to DNA, the conformational exchange contributions in R2 are reduced but remain significant in NMR relaxation measurements. Upon binding to DNA, the third helix of R3 comes to exhibit significant chemical exchange contributions. These findings suggest that the orientations of the third helices of both R2 and R3 as to DNA are being chemically exchanged. In the DNA-bound form both R2 and R3 exhibit similar dynamical characters, except for amino acids Trp 95, Thr 96, and Val 103 of R2, which are located around the cavity of the unbound form. Upon binding to DNA, since Trp 95 moves into the cavity to fill it up, the local conformational exchange contributions seem to be still observable around the filled cavity.
c-Myb的DNA结合结构域由三个不完全的串联重复序列R1、R2和R3组成。每个重复序列包含三个螺旋。最小的DNA结合结构域是一个R2R3片段。在此，我们通过核磁共振研究了R2R3与DNA结合形式下的主链动力学。与DNA结合后，N端和C端以及R2和R3之间的连接区变得不那么灵活。在游离形式下，R2的第三个螺旋由于R2疏水核心中的一个空洞而表现出缓慢的构象交换波动。与DNA结合后，R2中的构象交换贡献减少，但在核磁共振弛豫测量中仍然显著。与DNA结合后，R3的第三个螺旋开始表现出显著的化学交换贡献。这些发现表明，R2和R3的第三个螺旋相对于DNA的取向正在进行化学交换。在与DNA结合的形式中，R2和R3都表现出相似的动力学特征，除了R2的氨基酸色氨酸95、苏氨酸96和缬氨酸103，它们位于未结合形式的空洞周围。与DNA结合后，由于色氨酸95移入空洞以填充它，在填充的空洞周围似乎仍然可以观察到局部构象交换贡献。
