Crystal structure and functional analysis of DEAD-box protein Dhh1p
Crystal structure and functional analysis of DEAD-box protein Dhh1p
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作者：
Cheng, ZH;Coller, J;Song, H
控制信使核糖核酸的翻译和降解是基因正确表达的关键。翻译和降解的关键调节因子是Dhh1p，它是一种死盒蛋白，既能抑制翻译，又能促进解脱。我们描述了在2.1A分辨率下测定的N端和C端截断的Dhh1p(TDhh1p)的晶体结构。这表明，像其他死盒蛋白一样，tDhh1p包含两个类似RecA的结构域，尽管有独特的排列。与eIF4A和mjDEAD不存在基序相互作用相反，在Dhh1p中，基序V与基序I和Q-基序相互作用，从而将这两个结构域连接在一起。静电电位作图与突变相结合表明，基序1、V和VI参与了RNA的结合。此外，tDhh1p的胰酶消化表明，ATP结合增强了RNA诱导的构象变化。有趣的是，一些位于保守基序和两个Dhh1结构域之间的突变在体内提供了显性的负表型，并在体外破坏了构象转换。这表明这种构象变化是Dhh1功能所必需的，并确定了参与这一转换的关键残基。
The control of mRNA translation and degradation are critical for proper gene expression. A key regulator of both translation and degradation is Dhh1p, which is a DEAD-box protein, and functions both to repress translation and enhance decapping. We describe the crystal structure of the N- and C-terminal truncated Dhh1p (tDhh1p) determined at 2.1 A resolution. This reveals that, like other DEAD-box proteins, tDhh1p contains two RecA-like domains, although with a unique arrangement. In contrast to eIF4A and mjDEAD, in which no motif interactions exist, in Dhh1p, motif V interacts with motif I and the Q-motif, thereby linking the two domains together. Electrostatic potential mapping combined with mutagenesis reveals that motifs 1, V, and VI are involved in RNA binding. In addition, trypsin digestion of tDhh1p suggests that ATP binding enhances an RNA-induced conformational change. Interestingly, some mutations located in the conserved motifs and at the interface between the two Dhh1 domains confer dominant negative phenotypes in vivo and disrupt the conformational switch in vitro. This suggests that this conformational change is required in Dhh1 function and identifies key residues involved in that transition.