Center for Molecular Protein Science, Lund University, Getingevägen 60, 221 00, Lund, Sweden.
Biophys Chem. 2009 Dec;145(2-3):116-25. doi: 10.1016/j.bpc.2009.09.009. Epub 2009 Oct 1.
Wilms Tumor suppressor protein (WT1) is a transcription factor that is involved in a variety of developmental functions during organ development. It is also implicated in the pathology of several different cancer forms. The protein contains four C(2)H(2)-type zinc fingers and it specifically binds GC-rich sequences in the promoter regions of its target genes, which are either up or down regulated. Two properties make WT1 a more unusual transcription factor - an unconventional amino acid composition for zinc finger 1, and the insertion of a tri-peptide KTS in some of the splice isoforms of WT1. Using six WT1 constructs in which zinc fingers are systematically deleted, a dual study based on a bacterial 1-hybrid system and surface plasmon resonance measurements is performed. The experiments show that the effect of zinc finger 1 is not significant in terms of overall DNA-binding kinetics, however it influences both the specificity of target recognition and stability of interaction in presence of KTS. The KTS insertion, however, only mildly retards binding affinity, mainly by affecting the on-rate. We suggest that the insertion disturbs zinc finger 4 from its binding frame, thus weakening the rate of target recognition. Finally, for the construct in which both zinc fingers 1 and 4 were deleted, the two middle fingers 2-3 still could function as a 'minimal DNA-recognition domain' for WT1, however the formation of a stable protein-DNA complex is impaired since the overall affinity was dramatically reduced mainly since the off-rate was severely affected.
Wilms 肿瘤抑制蛋白（WT1）是一种转录因子，参与器官发育过程中的多种发育功能。它也与几种不同癌症形式的病理学有关。该蛋白包含四个 C(2)H(2)- 型锌指，它特异性地结合其靶基因启动子区域中的 GC 富集序列，这些序列被上调或下调。WT1 作为一种不同寻常的转录因子有两个特性——锌指 1 的非常规氨基酸组成，以及 WT1 的一些剪接异构体中插入三肽 KTS。使用六个系统删除锌指的 WT1 构建体，在细菌 1 杂交系统和表面等离子体共振测量的基础上进行了双重研究。实验表明，锌指 1 的作用在总体 DNA 结合动力学方面并不显著，但它影响靶识别的特异性和 KTS 存在时相互作用的稳定性。然而，KTS 的插入仅轻微降低结合亲和力，主要通过影响进入速率。我们认为插入会干扰锌指 4 与其结合框架的结合，从而削弱靶识别的速度。最后，对于同时删除锌指 1 和 4 的构建体，中间的两个锌指 2-3 仍然可以作为 WT1 的“最小 DNA 识别结构域”，但是由于整体亲和力显著降低，主要是由于退出速率受到严重影响，因此稳定的蛋白质-DNA 复合物的形成受到损害。
