Department of Biological Sciences and Centre for Bioimaging Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore.
Mol Plant. 2013 Jul;6(4):1290-300. doi: 10.1093/mp/sst007. Epub 2013 Jan 11.
Dawdle (DDL) is a microRNA processing protein essential for the development of Arabidopsis. DDL contains a putative nuclear localization signal at its amino-terminus and forkhead-associated (FHA) domain at the carboxyl-terminus. Here, we report the crystal structure of the FHA domain of Arabidopsis Dawdle, determined by multiple-wavelength anomalous dispersion method at 1.7-Å resolution. DDL FHA structure displays a seven-stranded β-sandwich architecture that contains a unique structural motif comprising two long anti-parallel strands. Strikingly, crystal packing of the DDL FHA domain reveals that a glutamate residue from the symmetry-related DDL FHA domain, a structural mimic of the phospho-threonine, is specifically recognized by the structurally conserved phospho-threonine binding cleft. Consistently with the structural observations, co-immuno-precipitation experiments performed in Nicotiana benthamiana show that the DDL FHA domain co-immuno-precipitates with DCL1 fragments containing the predicted pThr+3(Ile/Val/Leu/Asp) motif. Taken together, we count the recognition of the target residue by the canonical binding cleft of the DDL FHA domain as the key molecular event to instate FHA domain-mediated protein-protein interaction in plant miRNA processing.
拖拉（DDL）是一种 miRNA 加工蛋白，对拟南芥的发育至关重要。DDL 在其氨基末端包含一个假定的核定位信号，在羧基末端包含一个 forkhead 相关（FHA）结构域。在这里，我们通过多波长异常分散法在 1.7-Å 分辨率下报告了拟南芥 DDL 的 FHA 结构域的晶体结构。DDL FHA 结构显示出一个由七个β-折叠组成的三明治结构，其中包含一个独特的结构基序，包括两个长的反平行链。引人注目的是，DDL FHA 结构域的晶体堆积表明，来自对称相关的 DDL FHA 结构域的谷氨酸残基，是磷酸苏氨酸的结构模拟物，被结构保守的磷酸苏氨酸结合裂隙特异性识别。与结构观察一致，在 Nicotiana benthamiana 中进行的共免疫沉淀实验表明，DDL FHA 结构域与包含预测的 pThr+3（Ile/Val/Leu/Asp）基序的 DCL1 片段共免疫沉淀。总之，我们认为 DDL FHA 结构域的靶残基通过典型结合裂隙的识别是启动植物 miRNA 加工中 FHA 结构域介导的蛋白质-蛋白质相互作用的关键分子事件。
