The amino-terminal domain of human STAT4 - Overproduction, purification, and biophysical characterization
The amino-terminal domain of human STAT4 - Overproduction, purification, and biophysical characterization
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DOI：
10.1074/jbc.273.27.17109
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发表时间：
1998-07-03
影响因子：
4.8
通讯作者：
Waugh, DS
中科院分区：
文献类型：
--
作者：
Baden, HA;Sarma, SP;Waugh, DS
多功能信号转导和转录激活因子（STAT）蛋白在细胞因子和生长因子的作用下将信号从细胞膜传递到细胞核。当细胞被白细胞介素-12（细胞介导免疫的关键细胞因子调节剂）处理时，STAT4被激活。激活后，STAT4二聚体协同结合干扰素γ基因附近的串联干扰素γ激活序列（GAS元件）并刺激其转录。STAT4的氨基末端结构域（STAT4(1-124)）是STAT4二聚体之间的协同结合相互作用和干扰素- γ转录激活在响应白介素-12时所必需的。我们在大肠杆菌中过量生产了人类STAT4的这个结构域（hSTAT4(1-124)），并将其纯化到均匀性，用于结构研究。hSTAT4（1-124）的圆二色光谱表明其在溶液中具有良好的有序构象。通过核磁共振方法测定了hSTAT4（1-124）的平移扩散常数，发现其与二聚体的平移扩散常数一致。从N-15弛豫估计hSTAT4（1-124）的旋转相关时间（tau(c)）为16 ns；该值与29kda二聚体蛋白一致。这些结果，以及在均匀n -15标记蛋白的二维H-1-N-15异核单量子相干谱中观察到的信号数量，表明hSTAT4（1-124）在溶液中形成稳定的对称同型二聚体。原生STAT4的协同作用可能是由与DNA结合的相邻二聚体的氨基末端结构域之间类似或相同的相互作用引起的。
The multifunctional signal transducer and activator of transcription (STAT) proteins relay signals from the cell membrane to the nucleus in response to cytokines and growth factors. STAT4 becomes activated when cells are treated with interleukin-12, a key cytokine regulator of cell-mediated immunity. Upon activation, dimers of STAT4 bind cooperatively to tandem interferon-gamma activation sequences (GAS elements) near the interferon-gamma gene and stimulate its transcription. The amino-terminal domain of STAT4 (STAT4(1-124)) is required for cooperative binding interactions between STAT4 dimers and activation of interferon-gamma transcription in response to interleukin-12. me have overproduced this domain of human STAT4 (hSTAT4(1-124)) in Escherichia coli and purified it to homogeneity for structural studies. The circular dichroism spectrum of hSTAT4(1-124) indicates that it has a well ordered conformation in solution. The translational diffusion constant of hSTAT4(1-124) was determined by nuclear magnetic resonance methods and found to be consistent with that of a dimer. The rotational correlation time (tau(c)) of hSTAT4(1-124) was estimated from N-15 relaxation to be 16 ns; this value is consistent with a 29-kDa dimeric protein. These results, together with the number of signals observed in the two-dimensional H-1-N-15 heteronuclear single quantum coherence spectrum of uniformly N-15-labeled protein, indicate that hSTAT4(1-124) forms a stable, symmetric homodimer in solution. Cooperativity in native STAT4 probably results from a similar or identical interaction between the amino-terminal domains of adjacent dimers bound to DNA.