Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Protein Sci. 2012 Nov;21(11):1716-25. doi: 10.1002/pro.2151. Epub 2012 Oct 9.
Pointed-P2, the Drosophila ortholog of human ETS1 and ETS2, is a transcription factor involved in Ras/MAP kinase-regulated gene expression. In addition to a DNA-binding ETS domain, Pointed-P2 contains a PNT (or SAM) domain that serves as a docking module to enhance phosphorylation of an adjacent phosphoacceptor threonine by the ERK2 MAP kinase Rolled. Using NMR chemical shift, ¹⁵N relaxation, and amide hydrogen exchange measurements, we demonstrate that the Pointed-P2 PNT domain contains a dynamic N-terminal helix H0 appended to a core conserved five-helix bundle diagnostic of the SAM domain fold. Neither the secondary structure nor dynamics of the PNT domain is perturbed significantly upon in vitro ERK2 phosphorylation of three threonine residues in a disordered sequence immediately preceding this domain. These data thus confirm that the Drosophila Pointed-P2 PNT domain and phosphoacceptors are highly similar to those of the well-characterized human ETS1 transcription factor. NMR-monitored titrations also revealed that the phosphoacceptors and helix H0, as well as region of the core helical bundle identified previously by mutational analyses as a kinase docking site, are selectively perturbed upon ERK2 binding by Pointed-P2. Based on a homology model derived from the ETS1 PNT domain, helix H0 is predicted to partially occlude the docking interface. Therefore, this dynamic helix must be displaced to allow both docking of the kinase, as well as binding of Mae, a Drosophila protein that negatively regulates Pointed-P2 by competing with the kinase for its docking site.
果蝇直系同源物 Pointed-P2 是一种转录因子，与人类 ETS1 和 ETS2 同源，参与 Ras/MAP 激酶调控的基因表达。Pointed-P2 除了具有 DNA 结合 ETS 结构域外，还含有一个 PNT（或 SAM）结构域，作为一个停泊模块，增强 ERK2 MAP 激酶 Rolled 对相邻磷酸受体苏氨酸的磷酸化。利用核磁共振化学位移、¹⁵N 弛豫和酰胺氢交换测量，我们证明 Pointed-P2 PNT 结构域包含一个动态的 N 端螺旋 H0，连接到一个核心保守的五螺旋束，该结构束是 SAM 结构域折叠的特征。在体外 ERK2 对该结构域前面无序序列中三个苏氨酸残基的磷酸化作用下，PNT 结构域的二级结构和动力学均未受到显著影响。这些数据证实，果蝇 Pointed-P2 PNT 结构域和磷酸受体与经过充分研究的人 ETS1 转录因子非常相似。NMR 监测滴定还表明，磷酸受体和螺旋 H0，以及以前通过突变分析鉴定为激酶停泊位点的核心螺旋束区域，在 ERK2 结合 Pointed-P2 时会被选择性地扰乱。基于来自 ETS1 PNT 结构域的同源模型，预测螺旋 H0 会部分遮挡停泊界面。因此，这个动态螺旋必须被移位，以便激酶能够停泊，同时也允许果蝇蛋白 Mae 结合，Mae 通过与激酶竞争停泊位点来负调控 Pointed-P2。
