Doucleff Michaeleen, Chen Baoyu, Maris Ann E, Wemmer David E, Kondrashkina Elena, Nixon B Tracy
Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
J Mol Biol. 2005 Oct 21;353(2):242-55. doi: 10.1016/j.jmb.2005.08.003.
Only a few transcriptional regulatory proteins have been characterized in extremely hyperthermophilic organisms, and most function as repressors. Structural features of the NtrC1 protein from the hyperthermophilic bacterium Aquifex aeolicus suggested that this protein functions similarly to the sigma(54)-polymerase activator DctD of Sinorhizobium meliloti. Here, we demonstrate that NtrC1 is an enzyme that hydrolyzes ATP to activate initiation of transcription by sigma(54)-holoenzyme. New structural data, including small-angle solution scattering data and the crystal structure of the phosphorylated receiver domain, show that NtrC1 uses a signal transduction mechanism very similar to that of DctD to control assembly of its AAA+ ATPase domain. As for DctD, the off-state of NtrC1 depends upon a tight dimer of the receiver domain to repress oligomerization of an intrinsically competent ATPase domain. Activation of NtrC1 stabilizes an alternative dimer configuration of the receiver domain that is very similar to the on-state dimers of the DctD and FixJ receiver domains. This alternative dimer appears to relieve repression of the ATPase domain by disrupting the off-state dimerization interface along the helical linker region between receiver and ATPase domains. Bacterial enhancer binding proteins typically have two linker sequences, one between N-terminal regulatory and central ATPase domains, and one between the central ATPase and C-terminal DNA binding domains. Sequence analyses reveal an intriguing correlation between the negative regulation mechanism of NtrC1 and DctD, and a structured N-terminal linker and unstructured C-terminal one; conversely, the very different, positive mechanism present in NtrC protein occurs in the context of an unstructured N-terminal linker and a structured C-terminal one. In both cases, the structured linkers significantly contribute to the stability of the off-state dimer conformation. These analyses also raise the possibility that a structured linker between N-terminal regulatory and central output domains is used frequently in regulatory proteins from hyperthermophilic organisms.
在极端嗜热生物中，仅有少数转录调节蛋白得到了表征，且大多数发挥阻遏物的作用。嗜热细菌嗜热栖热菌（Aquifex aeolicus）的NtrC1蛋白的结构特征表明，该蛋白的功能与苜蓿中华根瘤菌（Sinorhizobium meliloti）的σ(54)-聚合酶激活剂DctD相似。在此，我们证明NtrC1是一种水解ATP以激活σ(54)-全酶转录起始的酶。新的结构数据，包括小角溶液散射数据和磷酸化受体结构域的晶体结构，表明NtrC1利用一种与DctD非常相似的信号转导机制来控制其AAA+ ATP酶结构域的组装。与DctD一样，NtrC1的关闭状态依赖于受体结构域的紧密二聚体来抑制固有活性的ATP酶结构域的寡聚化。NtrC1的激活稳定了受体结构域的另一种二聚体构型，该构型与DctD和FixJ受体结构域的开启状态二聚体非常相似。这种替代二聚体似乎通过破坏受体和ATP酶结构域之间螺旋连接区域的关闭状态二聚化界面来解除对ATP酶结构域的抑制。细菌增强子结合蛋白通常有两个连接序列，一个在N端调节结构域和中央ATP酶结构域之间，另一个在中央ATP酶结构域和C端DNA结合结构域之间。序列分析揭示了NtrC1和DctD的负调控机制与结构化的N端连接子和非结构化的C端连接子之间有趣的相关性；相反，NtrC蛋白中非常不同的正调控机制则发生在非结构化的N端连接子和结构化的C端连接子的背景下。在这两种情况下，结构化连接子都对关闭状态二聚体构象的稳定性有显著贡献。这些分析还提出了一种可能性，即N端调节结构域和中央输出结构域之间的结构化连接子在嗜热生物的调节蛋白中经常被使用。
