Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA 94158, USA.
Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164, USA.
J Mol Biol. 2014 May 15;426(10):2034-44. doi: 10.1016/j.jmb.2014.02.021. Epub 2014 Mar 4.
The chromatin remodeling complex ACF helps establish the appropriate nucleosome spacing for generating repressed chromatin states. ACF activity is stimulated by two defining features of the nucleosomal substrate: a basic patch on the histone H4 N-terminal tail and the specific length of flanking DNA. However, the mechanisms by which these two substrate cues function in the ACF remodeling reaction is not well understood. Using electron paramagnetic resonance spectroscopy with spin-labeled ATP analogs to probe the structure of the ATP active site under physiological solution conditions, we identify a closed state of the ATP-binding pocket that correlates with ATPase activity. We find that the H4 tail promotes pocket closure. We further show that ATPase stimulation by the H4 tail does not require a specific structure connecting the H4 tail and the globular domain. In the case of many DNA helicases, closure of the ATP-binding pocket is regulated by specific DNA substrates. Pocket closure by the H4 tail may analogously provide a mechanism to directly couple substrate recognition to activity. Surprisingly, the flanking DNA, which also stimulates ATP hydrolysis, does not promote pocket closure, suggesting that the H4 tail and flanking DNA may be recognized in different reaction steps.
染色质重塑复合物 ACF 有助于为产生受抑制的染色质状态建立适当的核小体间距。ACF 活性受到核小体底物两个定义特征的刺激：组蛋白 H4 N 端尾巴上的碱性斑和侧翼 DNA 的特定长度。然而，这两个底物线索在 ACF 重塑反应中起作用的机制尚不清楚。我们使用带有自旋标记的 ATP 类似物的电子顺磁共振波谱法在生理溶液条件下探测 ATP 活性位点的结构，确定与 ATP 酶活性相关的 ATP 结合口袋的封闭状态。我们发现 H4 尾巴促进口袋关闭。我们进一步表明，H4 尾巴对 ATP 酶的刺激不需要连接 H4 尾巴和球形结构域的特定结构。对于许多 DNA 解旋酶，ATP 结合口袋的闭合受特定 DNA 底物的调节。H4 尾巴的口袋闭合可能类似地提供了一种将底物识别与活性直接偶联的机制。令人惊讶的是，同样刺激 ATP 水解的侧翼 DNA不会促进口袋闭合，这表明 H4 尾巴和侧翼 DNA 可能在不同的反应步骤中被识别。
