Structural Biology and NMR Laboratory, Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark.
Computational Biology Laboratory, Center for Autophagy, Recycling and Disease, Strandboulevarden 49, 2100 Copenhagen, Denmark.
Cell Signal. 2017 Sep;37:40-51. doi: 10.1016/j.cellsig.2017.05.015. Epub 2017 May 26.
Intrinsically disordered proteins (IDPs) are involved in many pivotal cellular processes including phosphorylation and signalling. The structural and functional effects of phosphorylation of IDPs remain poorly understood and difficult to predict. Thus, a need exists to identify motifs that confer phosphorylation-dependent perturbation of the local preferences for forming e.g. helical structures as well as motifs that do not. The disordered distal tail of the Na/H exchanger 1 (NHE1) is six-times phosphorylated (S693, S723, S726, S771, T779, S785) by the mitogen activated protein kinase 2 (MAPK1, ERK2). Using NMR spectroscopy, we found that two out of those six phosphorylation sites had a stabilizing effect on transient helices. One of these was further investigated by circular dichroism and NMR spectroscopy as well as by molecular dynamic simulations, which confirmed the stabilizing effect and resulted in the identification of a short linear motif for helix stabilisation: [S/T]-P-{3}-[R/K] where [S/T] is the phosphorylation-site. By analysing IDP and phosphorylation site databases we found that the motif is significantly enriched around known phosphorylation sites, supporting a potential wider-spread role in phosphorylation-mediated regulation of intrinsically disordered proteins. The identification of such motifs is important for understanding the molecular mechanism of cellular signalling, and is crucial for the development of predictors for the structural effect of phosphorylation; a tool of relevance for understanding disease-promoting mutations that for example interfere with signalling for instance through constitutive active and often cancer-promoting signalling.
无规则蛋白质（IDPs）参与许多关键的细胞过程，包括磷酸化和信号转导。IDPs 磷酸化的结构和功能影响仍然知之甚少，难以预测。因此，需要确定能够赋予磷酸化依赖性扰动的基序，例如形成螺旋结构的局部偏好的基序，以及不赋予这种扰动的基序。钠/氢交换器 1（NHE1）的无规则远末端被丝裂原活化蛋白激酶 2（MAPK1，ERK2）磷酸化 6 次（S693、S723、S726、S771、T779、S785）。使用 NMR 光谱，我们发现这 6 个磷酸化位点中有 2 个对瞬时螺旋有稳定作用。其中一个进一步通过圆二色性和 NMR 光谱以及分子动力学模拟进行了研究，这证实了稳定作用，并确定了一个用于螺旋稳定的短线性基序：[S/T]-P-{3}-[R/K]，其中 [S/T] 是磷酸化位点。通过分析 IDP 和磷酸化位点数据库，我们发现该基序在已知磷酸化位点周围显著富集，这支持了它在磷酸化介导的无规则蛋白质调节中更广泛的作用。这种基序的鉴定对于理解细胞信号转导的分子机制很重要，对于开发用于预测磷酸化对结构影响的预测器也很关键；这是一种用于理解促进疾病的突变的工具，例如通过组成性激活和经常促进癌症的信号转导来干扰信号转导。
