Structural Regulation of a Neurofilament-Inspired Intrinsically Disordered Protein Brush by Multisite Phosphorylation.
Structural Regulation of a Neurofilament-Inspired Intrinsically Disordered Protein Brush by Multisite Phosphorylation.
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DOI：
10.1021/acs.biochem.8b00007
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发表时间：
2018-03
期刊：
影响因子：
2.9
通讯作者：
Ruoxing Lei;Jess P Lee;M. Francis;Sanjay Kumar
中科院分区：
文献类型：
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作者：
Ruoxing Lei;Jess P Lee;M. Francis;Sanjay Kumar
本质无序蛋白 (IDP) 在许多细胞过程中发挥着核心作用。虽然 IDP 结构和功能通常受到多位点磷酸化的调节，但将这些翻译后修饰与 IDP 结构联系起来的生物物理机制仍然难以捉摸。例如，神经丝重亚基 (NFH-SA) 本质上无序的 C 端侧臂结构域沿着轴突 NF 骨干形成密集的刷子，并受到广泛的丝氨酸磷酸化。然而，由于缺乏可以在受控磷酸化环境中测量 NF 刷构象反应的范例，对磷酸化和结构之间关系的生物物理学洞察受到限制。在这里，我们通过将重组 NFH-SA (rNFH-SA) 固定为 IDP 刷到玻璃上来解决这个问题，并用丝裂原激活蛋白激酶激酶 (MKK) 预激活的丝裂原激活蛋白激酶 1 (ERK2) 控制原位磷酸化序列。然后，我们使用原子力显微镜监测刷子高度变化，结果表明磷酸化会引起显着的刷子膨胀，其程度强烈依赖于pH和离子强度，这与磷酸化通过局部静电相互作用调节刷子结构的机制一致。与该机制进一步一致的是，磷酸化的rNFH-SA刷可能与微摩尔浓度的二价阳离子显着缩合。磷酸化引起的高度变化通过碱性磷酸酶介导的去磷酸化是可逆的。我们的研究表明，多位点磷酸化通过调节链静电来控制 NFH-SA 结构，并指出了工程基于 IDP 的界面的一般策略，该界面可以通过酶进行可逆和动态调节。
Intrinsically disordered proteins (IDPs) play central roles in numerous cellular processes. While IDP structure and function are often regulated by multisite phosphorylation, the biophysical mechanisms linking these post-translational modifications to IDP structure remain elusive. For example, the intrinsically disordered C-terminal sidearm domain of the neurofilament heavy subunit (NFH-SA) forms a dense brush along axonal NF backbones and is subject to extensive serine phosphorylation. Yet, biophysical insight into the relationship between phosphorylation and structure has been limited by the lack of paradigms in which NF brush conformational responses can be measured in the setting of controlled phosphorylation. Here, we approach this question by immobilizing a recombinant NFH-SA (rNFH-SA) as IDP brushes onto glass, and controllably phosphorylating the sequence in situ with mitogen-activated protein kinase 1 (ERK2) preactivated by mitogen-activated protein kinase kinase (MKK). We then monitor brush height changes using atomic force microscopy, which shows that phosphorylation induces significant brush swelling to an extent that strongly depends upon pH and ionic strength, consistent with a mechanism in which phosphorylation regulates brush structure through local electrostatic interactions. Further consistent with this mechanism, the phosphorylated rNFH-SA brush may be dramatically condensed with micromolar concentrations of divalent cations. Phosphorylation-induced height changes are qualitatively reversible via alkaline phosphatase-mediated dephosphorylation. Our study demonstrates that multisite phosphorylation controls NFH-SA structure through modulation of chain electrostatics and points to a general strategy for engineering IDP-based interfaces that can be reversibly and dynamically modulated by enzymes.