Autophosphorylation of Archaeoglobus fulgidus Rio2 and crystal structures of its nucleotide-metal ion complexes
Autophosphorylation of Archaeoglobus fulgidus Rio2 and crystal structures of its nucleotide-metal ion complexes
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DOI：
10.1111/j.1742-4658.2005.04702.x
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发表时间：
2005-06-01
期刊：
影响因子：
5.4
通讯作者：
Wlodawer, A
中科院分区：
文献类型：
--
作者：
LaRonde-LeBlanc, N;Guszczynski, T;Wlodawer, A
高度保守的非典型RIO丝氨酸蛋白激酶存在于从古细菌到人类的所有生物体中。在酵母中，RIO 2的激酶活性对于18 S核糖体rRNA成熟的最后加工步骤是必需的。我们以前已经表明，从闪烁古生球菌Rio 2蛋白包含一个小的激酶结构域和一个N-末端翅螺旋结构域。先前使用浸泡在核苷酸和Mg 2+或Mn 2+中的晶体解决的结构显示结合的核苷酸，但没有有序的金属离子，这使我们得出结论，它们不代表酶的活性构象。为了确定Rio 2的功能形式，我们在与ATP或ADP和Mn 2+孵育后使其结晶。分别在1.84和1.75埃分辨率下解析R102-ATP-Mn和R102-ADP-Mn的共晶结构。ATP的γ-磷酸以与其在经典丝氨酸激酶中的位置明显不同的方式牢固地定位。R102-ATP-Mn复合物与不添加核苷酸的R102结构和与ADP复合物的比较表明，R102分子的柔性部分通过His 126和ATP的γ-磷酸氧之间的直接相互作用而变得有序。R102的自磷酸化位点的磷酸肽图谱确定了Ser 128，其位于柔性环内，并直接邻近响应ATP而变得有序的部分，作为靶标。这些结果为我们提供了关于Rio 2激酶活性位点的性质的进一步信息，并提出了其酶活性的调节机制。
The highly conserved, atypical RIO serine protein kinases are found in all organisms, from archaea to man. In yeast, the kinase activity of Rio2 is necessary for the final processing step of maturing the 18S ribosomal rRNA. We have previously shown that the Rio2 protein from Archaeoglobus fulgidus contains both a small kinase domain and an N-terminal winged helix domain. Previously solved structures using crystals soaked in nucleotides and Mg2+ or Mn2+ showed bound nucleotide but no ordered metal ions, leading us to the conclusion that they did not represent an active conformation of the enzyme. To determine the functional form of Rio2, we crystallized it after incubation with ATP or ADP and Mn2+. Co-crystal structures of Rio2-ATP-Mn and Rio2-ADP-Mn were solved at 1.84 and 1.75 angstrom resolution, respectively. The gamma-phosphate of ATP is firmly positioned in a manner clearly distinct from its location in canonical serine kinases. Comparison of the Rio2-ATP-Mn complex with the Rio2 structure with no added nucleotides and with the ADP complex indicates that a flexible portion of the Rio2 molecule becomes ordered through direct interaction between His126 and the gamma-phosphate oxygen of ATP. Phosphopeptide mapping of the autophosphorylation site of Rio2 identified Ser128, within the flexible loop and directly adjacent to the part that becomes ordered in response to ATP, as the target. These results give us further information about the nature of the active site of Rio2 kinase and suggest a mechanism of regulation of its enzymatic activity.