Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.
Department of Molecular and Cellular Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
Nucleic Acids Res. 2018 May 4;46(8):3864-3877. doi: 10.1093/nar/gkx1274.
Wilms tumor protein (WT1) is a Cys2-His2 zinc-finger transcription factor vital for embryonic development of the genitourinary system. The protein contains a C-terminal DNA binding domain with four tandem zinc-fingers (ZF1-4). An alternative splicing of Wt1 can add three additional amino acids-lysine (K), threonine (T) and serine (S)-between ZF3 and ZF4. In the -KTS isoform, ZF2-4 determine the sequence-specificity of DNA binding, whereas the function of ZF1 remains elusive. Three X-ray structures are described here for wild-type -KTS isoform ZF1-4 in complex with its cognate DNA sequence. We observed four unique ZF1 conformations. First, like ZF2-4, ZF1 can be positioned continuously in the DNA major groove forming a 'near-cognate' complex. Second, while ZF2-4 make base-specific interactions with one DNA molecule, ZF1 can interact with a second DNA molecule (or, presumably, two regions of the same DNA molecule). Third, ZF1 can intercalate at the joint of two tail-to-head DNA molecules. If such intercalation occurs on a continuous DNA molecule, it would kink the DNA at the ZF1 binding site. Fourth, two ZF1 units can dimerize. Furthermore, we examined a Denys-Drash syndrome-associated ZF1 mutation (methionine at position 342 is replaced by arginine). This mutation enhances WT1 affinity for a guanine base. X-ray crystallography of the mutant in complex with its preferred sequence revealed the interactions responsible for this affinity change. These results provide insight into the mechanisms of action of WT1, and clarify the fact that ZF1 plays a role in determining sequence specificity of this critical transcription factor.
Wilms 肿瘤蛋白（WT1）是一种 Cys2-His2 锌指转录因子，对生殖泌尿系统的胚胎发育至关重要。该蛋白含有一个 C 端 DNA 结合域，其中包含四个串联锌指（ZF1-4）。Wt1 的选择性剪接可以在 ZF3 和 ZF4 之间添加三个额外的氨基酸-赖氨酸（K）、苏氨酸（T）和丝氨酸（S）。在-KTS 异构体中，ZF2-4 决定 DNA 结合的序列特异性，而 ZF1 的功能仍不清楚。本文描述了野生型-KTS 异构体 ZF1-4 与同源 DNA 序列形成复合物的四个独特的 ZF1 构象。首先，与 ZF2-4 一样，ZF1 可以连续定位在 DNA 大沟中，形成“近同源”复合物。其次，虽然 ZF2-4 与一个 DNA 分子进行碱基特异性相互作用，但 ZF1 可以与第二个 DNA 分子（或者，大概是同一 DNA 分子的两个区域）相互作用。第三，ZF1 可以在两个尾巴对头 DNA 分子的连接处插入。如果这种插入发生在连续的 DNA 分子上，它会使 DNA 在 ZF1 结合位点处扭曲。第四，两个 ZF1 单元可以二聚化。此外，我们研究了与 Denys-Drash 综合征相关的 ZF1 突变（第 342 位的蛋氨酸被精氨酸取代）。这种突变增强了 WT1 对鸟嘌呤碱基的亲和力。突变体与首选序列形成复合物的 X 射线晶体学揭示了导致这种亲和力变化的相互作用。这些结果为 WT1 的作用机制提供了深入的了解，并澄清了 ZF1 在确定这个关键转录因子的序列特异性方面发挥作用的事实。
