Ewa Rejnowicz, Matthew Batchelor, Eoin Leen, Mohd Syed Ahangar, Selena G Burgess, Mark W Richards, Arnout P Kalverda, Richard Bayliss
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引用次数: 0
Abstract
Myc proteins are transcription factors crucial for cell proliferation. They have a C-terminal domain that mediates Max and DNA binding, and an N-terminal disordered region culminating in the transactivation domain (TAD). The TAD participates in many protein-protein interactions, notably with kinases that promote stability (Aurora-A) or degradation (ERK1, GSK3) via the ubiquitin-proteasome system. We probed the structure, dynamics and interactions of N-myc TAD using nuclear magnetic resonance (NMR) spectroscopy following its complete backbone assignment. Chemical shift analysis revealed that N-myc has two regions with clear helical propensity: Trp77-Glu86 and Ala122-Glu132. These regions also have more restricted ps-ns motions than the rest of the TAD, and, along with the phosphodegron, have comparatively high transverse (R2) 15N relaxation rates, indicative of slower timescale dynamics and/or chemical exchange. Collectively these features suggest differential propensities for structure and interaction, either internal or with binding partners, across the TAD. Solution studies on the interaction between N-myc and Aurora-A revealed a previously uncharacterised binding site. The specificity and kinetics of sequential phosphorylation of N-myc by ERK1 and GSK3 were characterised using NMR and resulted in no significant structural changes outside the phosphodegron. When the phosphodegron was doubly phosphorylated, N-myc formed a robust interaction with the Fbxw7-Skp1 complex, but mapping the interaction by NMR suggests a more extensive interface. Our study provides foundational insights into N-myc TAD dynamics and a backbone assignment that will underpin future work on the structure, dynamics, interactions and regulatory post-translational modifications of this key oncoprotein.
Myc 蛋白是对细胞增殖至关重要的转录因子。它们有一个介导 Max 和 DNA 结合的 C 端结构域,以及一个 N 端无序区,最终形成转录激活结构域(TAD)。TAD 参与了许多蛋白质与蛋白质之间的相互作用,特别是与通过泛素蛋白酶体系统促进稳定性(Aurora-A)或降解(ERK1、GSK3)的激酶的相互作用。我们利用核磁共振(NMR)光谱对 N-myc TAD 的结构、动力学和相互作用进行了探究,并对其骨架进行了完整的分配。化学位移分析表明,N-myc 有两个具有明显螺旋倾向的区域:Trp77-Glu86 和 Ala122-Glu132。与 TAD 的其他部分相比,这些区域的 ps-ns 运动也更受限制,而且与磷酸二聚体一起,具有相对较高的横向 (R2) 15N 松弛率,这表明时间尺度动力学和/或化学交换较慢。总之,这些特征表明整个 TAD 内部或与结合伙伴的结构和相互作用具有不同的倾向性。对 N-myc 和 Aurora-A 之间相互作用的溶液研究发现了一个以前未曾描述过的结合位点。利用核磁共振分析了 ERK1 和 GSK3 对 N-myc 顺序磷酸化的特异性和动力学特征,结果表明磷酸二聚体之外的结构没有发生显著变化。当磷酸化二聚体被双重磷酸化时,N-myc与Fbxw7-Skp1复合物形成了强有力的相互作用,但核磁共振绘制的相互作用图显示了一个更广泛的界面。我们的研究提供了有关 N-myc TAD 动力学和骨架分配的基本见解,这将为今后研究这种关键肿瘤蛋白的结构、动力学、相互作用和翻译后修饰调控提供基础。
期刊介绍:
Exploring the molecular mechanisms that underpin key biological processes, the Biochemical Journal is a leading bioscience journal publishing high-impact scientific research papers and reviews on the latest advances and new mechanistic concepts in the fields of biochemistry, cellular biosciences and molecular biology.
The Journal and its Editorial Board are committed to publishing work that provides a significant advance to current understanding or mechanistic insights; studies that go beyond observational work using in vitro and/or in vivo approaches are welcomed.
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