Research Department of Structural & Molecular Biology, University College London, London, United Kingdom.
PLoS One. 2013 Jul 29;8(7):e70095. doi: 10.1371/journal.pone.0070095. Print 2013.
Death-associated protein kinase (DAPk) is a calcium/calmodulin-regulated Ser/Thr-protein kinase that functions at an important point of integration for cell death signaling pathways. DAPk has a structurally unique multi-domain architecture, including a C-terminally positioned death domain (DD) that is a positive regulator of DAPk activity. In this study, recombinant DAPk-DD was observed to aggregate readily and could not be prepared in sufficient yield for structural analysis. However, DAPk-DD could be obtained as a soluble protein in the form of a translational fusion protein with the B1 domain of streptococcal protein G. In contrast to other DDs that adopt the canonical six amphipathic α-helices arranged in a compact fold, the DAPk-DD was found to possess surprisingly low regular secondary structure content and an absence of a stable globular fold, as determined by circular dichroism (CD), NMR spectroscopy and a temperature-dependent fluorescence assay. Furthermore, we measured the in vitro interaction between extracellular-regulated kinase-2 (ERK2) and various recombinant DAPk-DD constructs. Despite the low level of structural order, the recombinant DAPk-DD retained the ability to interact with ERK2 in a 1∶1 ratio with a K d in the low micromolar range. Only the full-length DAPk-DD could bind ERK2, indicating that the apparent 'D-motif' located in the putative sixth helix of DAPk-DD is not sufficient for ERK2 recognition. CD analysis revealed that binding of DAPk-DD to ERK2 is not accompanied by a significant change in secondary structure. Taken together our data argue that the DAPk-DD, when expressed in isolation, does not adopt a classical DD fold, yet in this state retains the capacity to interact with at least one of its binding partners. The lack of a stable globular structure for the DAPk-DD may reflect either that its folding would be supported by interactions absent in our experimental set-up, or a limitation in the structural bioinformatics assignment of the three-dimensional structure.
死亡相关蛋白激酶（DAPk）是一种钙/钙调蛋白调节的丝氨酸/苏氨酸蛋白激酶，在细胞死亡信号通路的整合点发挥重要作用。DAPk 具有独特的多结构域架构，包括位于 C 末端的死亡结构域（DD），它是 DAPk 活性的正调节剂。在这项研究中，观察到重组 DAPk-DD 容易聚集，并且无法以足够的产量制备用于结构分析。然而，DAPk-DD 可以作为与链球菌蛋白 G 的 B1 结构域的翻译融合蛋白的形式获得可溶性蛋白质。与采用紧凑折叠排列的典型六疏水 α-螺旋的其他 DD 不同，DAPk-DD 被发现具有惊人低的规则二级结构含量和缺乏稳定的球形折叠，这是通过圆二色性（CD）、NMR 光谱和温度依赖性荧光测定确定的。此外，我们测量了细胞外调节激酶-2（ERK2）与各种重组 DAPk-DD 构建体之间的体外相互作用。尽管结构有序性低，但重组 DAPk-DD 仍然能够以 1∶1 比例与 ERK2 相互作用，Kd 在低微摩尔范围内。只有全长 DAPk-DD 能够与 ERK2 结合，表明位于 DAPk-DD 假定第六螺旋中的明显“D 基序”不足以识别 ERK2。CD 分析表明，DAPk-DD 与 ERK2 的结合不会伴随二级结构的显著变化。总的来说，我们的数据表明，当单独表达时，DAPk-DD 不采用经典的 DD 折叠，但在这种状态下保留与至少一个结合伙伴相互作用的能力。DAPk-DD 缺乏稳定的球形结构可能反映了其折叠将由我们实验设置中不存在的相互作用支持，或者是结构生物信息学对三维结构的分配的限制。
