Solution structure determination of the two DNA-binding domains in the Shizosaccharomyces pombe Abp1 protein by a combination of dipolar coupling and diffusion anisotropy restraints
Solution structure determination of the two DNA-binding domains in the Shizosaccharomyces pombe Abp1 protein by a combination of dipolar coupling and diffusion anisotropy restraints
复制标题
通过偶极耦合和扩散各向异性约束相结合测定裂殖酵母 Abp1 蛋白中两个 DNA 结合域的溶液结构
DOI：
10.1023/a:1014977808170
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发表时间：
2002
影响因子：
2.7
通讯作者：
S. Yokoyama
中科院分区：
文献类型：
--
作者：
J. Kikuchi;J. Iwahara;T. Kigawa;Y. Murakami;T. Okazaki;S. Yokoyama
我们已经解决了分裂酵母着丝粒蛋白Abp1的N-末端区域的溶液结构，该蛋白质与带有其识别位点的21个碱基对的DNA片段结合(Mw=2.30 kDa)。虽然传统的基于NOE的核磁共振方法很好地定义了Abp1蛋白中的两个DNA结合域，但由于缺乏结构域之间的距离限制，Abp1蛋白的整体结构定义不够明确。因此，我们还使用了在弱排列状态下测量的剩余偶极耦合以及旋转扩散各向异性。由于光谱重叠，NH剩余偶极耦合和主干15N T1/T2数据都不足以确定Abp1蛋白的总体结构。我们结合使用了这两种取向约束(残余偶极耦合和旋转扩散各向异性)，显著改善了整体结构的收敛。观察到的t1/t2比(Abp1二级结构区的20-50)比以前报道的几种球状蛋白的数据要宽，表明Abp1·DNA复合体的整体形状是椭圆形的。这种扩展形式将有助于Apb1的DNA结合域识别相对较长的DNA序列中的两个单独的位置。
We have solved the solution structure of the N-terminal region of the fission yeast centromere protein, Abp1, bound to a 21-base pair DNA fragment bearing its recognition site (Mw = 30 kDa). Although the two DNA-binding domains in the Abp1 protein were defined well by a conventional NOE-based NMR methodology, the overall structure of the Abp1 protein was poorly defined, due to the lack of interdomain distance restraints. Therefore, we additionally used residual dipolar couplings measured in a weakly aligned state, and rotational diffusion anisotropies. Neither the NH residual dipolar couplings nor the backbone 15N T1/T2 data were sufficient to determine the overall structure of the Abp1 protein, due to spectral overlap. We used a combination of these two orientational restraints (residual dipolar coupling and rotational diffusion anisotropy), which significantly improved the convergence of the overall structures. The range of the observed T1/T2 ratios was wider (20–50 for the secondary structure regions of Abp1) than the previously reported data for several globular proteins, indicating that the overall shape of the Abp1•DNA complex is ellipsoid. This extended form would facilitate the recognition of the two separate sites in the relatively long DNA sequence by the DNA-binding domains of Apb1.