Laity J H, Chung J, Dyson H J, Wright P E
Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10596 North Torrey Pines Road, La Jolla, California 92037, USA.
Biochemistry. 2000 May 9;39(18):5341-8. doi: 10.1021/bi9926678.
The Wilms' tumor suppressor protein (WT1) is a zinc finger transcription factor that appears to function differently according to the presence of a posttranscriptional modification that adds three amino acids into one of the linker regions between the zinc fingers. We have investigated the structural consequences of the insertion of the Lys-Thr-Ser (KTS) sequence by preparing recombinant protein constructs of the four zinc finger DNA-binding domain of WT1 corresponding to the two isoforms with (+KTS) and without (-KTS) the insertion, which is located in the linker region between the third and fourth zinc fingers. NMR resonance assignments were used to estimate the structural differences between the two isoforms both free in solution and in complex with a 14 base pair DNA duplex corresponding to the WT1 recognition element. The NMR spectra indicate that the two isoforms are nearly identical in structure in the absence of the DNA. Only the immediate region of the insertion showed any change in chemical shifts. Upon DNA binding, the NMR spectrum of each isoform changed to indicate greater structure formation in the linker regions. Significant differences were observed between the spectra of the DNA complexes of the +KTS and -KTS isoforms, with the -KTS construct forming a more stable complex, consistent with prior biochemical assays. The majority of the differences between the spectra of the two complexes occur in the immediate region of the insertion, which appears to be closer in structure to the free form of the protein in the case of the +KTS complex. The insertion of the KTS sequence disrupts important interactions of the linker region with the adjacent zinc fingers, thus lowering the stability of the complex. The "normal" (-KTS) sequence of the linker appears to be involved in a C-terminal helix-capping interaction with the helix of the preceding zinc finger, a stabilizing interaction which is abrogated in the +KTS isoform.
威尔姆斯瘤抑制蛋白（WT1）是一种锌指转录因子，根据转录后修饰的存在情况，其功能似乎有所不同，这种修饰会在锌指之间的一个连接区添加三个氨基酸。我们通过制备WT1四个锌指DNA结合结构域的重组蛋白构建体来研究赖氨酸 - 苏氨酸 - 丝氨酸（KTS）序列插入的结构后果，该构建体对应于有（+KTS）和无（-KTS）插入的两种异构体，插入位于第三和第四锌指之间的连接区。核磁共振共振归属用于估计两种异构体在溶液中游离状态以及与对应WT1识别元件的14个碱基对DNA双链体形成复合物时的结构差异。核磁共振光谱表明，在没有DNA的情况下，两种异构体的结构几乎相同。只有插入的直接区域化学位移有任何变化。与DNA结合后，每种异构体的核磁共振光谱发生变化，表明连接区形成了更大的结构。在+KTS和 -KTS异构体的DNA复合物光谱之间观察到显著差异，-KTS构建体形成更稳定的复合物，这与先前的生化分析一致。两种复合物光谱之间的大部分差异出现在插入的直接区域，在+KTS复合物的情况下，该区域的结构似乎更接近蛋白质的游离形式。KTS序列的插入破坏了连接区与相邻锌指的重要相互作用，从而降低了复合物的稳定性。连接区的“正常”（-KTS）序列似乎参与了与前一个锌指螺旋的C端螺旋帽相互作用，这种稳定相互作用在+KTS异构体中被消除。
