Lei Ruoxing, Lee Jessica P, Francis Matthew B, Kumar Sanjay
Materials Sciences Division , Lawrence Berkeley National Laboratory , Berkeley , California 94720 , United States.
Biochemistry. 2018 Jul 10;57(27):4019-4028. doi: 10.1021/acs.biochem.8b00007. Epub 2018 Apr 6.
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.
内在无序蛋白（IDP）在众多细胞过程中发挥着核心作用。虽然IDP的结构和功能通常受多位点磷酸化调控，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不清楚。例如，神经丝重亚基（NFH）的内在无序C末端侧臂结构域（NFH-SA）沿着轴突NF主干形成密集的刷状结构，并经历广泛的丝氨酸磷酸化。然而，由于缺乏能够在可控磷酸化条件下测量NF刷构象反应的范例，对磷酸化与结构之间关系的生物物理理解一直受到限制。在这里，我们通过将重组NFH-SA（rNFH-SA）作为IDP刷固定在玻璃上，并使用丝裂原活化蛋白激酶激酶（MKK）预激活的丝裂原活化蛋白激酶1（ERK2）原位可控地磷酸化该序列来解决这个问题。然后，我们使用原子力显微镜监测刷高度的变化，结果表明磷酸化会诱导显著的刷膨胀，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刷可能会被微摩尔浓度的二价阳离子显著浓缩。磷酸化诱导的高度变化可通过碱性磷酸酶介导的去磷酸化在质量上逆转。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的结构，并指出了一种工程化基于IDP的界面的通用策略，该界面可由酶进行可逆和动态调节。
