Insights into Phosphorylation-Induced Protein Allostery and Conformational Dynamics of Glycogen Phosphorylase via Integrative Structural Mass Spectrometry and In Silico Modeling
Insights into Phosphorylation-Induced Protein Allostery and Conformational Dynamics of Glycogen Phosphorylase via Integrative Structural Mass Spectrometry and In Silico Modeling
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通过综合结构质谱和计算机模拟深入了解磷酸化诱导的蛋白质变构和糖原磷酸化酶的构象动力学
DOI：
10.1021/acschembio.2c00393
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发表时间：
2022-06-08
影响因子：
4
通讯作者：
Li, Huilin
中科院分区：
文献类型：
--
作者：
Huang, Jing;Chu, Xiakun;Li, Huilin
变构调节在无数生物过程中起着重要作用。在分子水平上了解其动力学机制和影响对于疾病诊断和药物发现具有重要意义。糖原磷酸化酶（GP）是一种受别构调节的磷蛋白，对糖原代谢具有重要的生物学意义。虽然GP的原子结构以前已经解决，与变构调节相关的GP的构象动力学由于其大分子大小（类似于196 kDa）而在很大程度上仍然难以捉摸。在这里，我们集成了原生自上而下的质谱（nTD-MS），氢氘交换MS（HDX-MS），保护因子（PF）分析，分子动力学（MD）模拟，和变构信号分析，以检查磷酸化的GP的变构调节的结构基础和动力学。nTD-MS揭示了非磷酸化（GPb）和磷酸化（GPa）形式之间的结构稳定性以及寡聚状态的差异。HDX-MS，PF分析和MD模拟进一步查明GPb和GPa之间的结构差异，涉及结合界面（N-末端和塔-塔螺旋），催化位点和PLP结合区域。更重要的是，它还让我们完成了由磷酸化引起的从N端尾部到催化位点的远程通信过程中缺失的一环。这种集成的MS和基于计算机的平台与生物物理方法高度互补，并对蛋白质结构和动态调节产生有价值的见解。
Allosteric regulation plays a fundamental role in innumerable biological processes. Understanding its dynamic mechanism and impact at the molecular level is of great importance in disease diagnosis and drug discovery. Glycogen phosphorylase (GP) is a phosphoprotein responding to allosteric regulation and has significant biological importance to glycogen metabolism. Although the atomic structures of GP have been previously solved, the conformational dynamics of GP related to allostery regulation remain largely elusive due to its macromolecular size (similar to 196 kDa). Here, we integrated native top-down mass spectrometry (nTD-MS), hydrogen-deuterium exchange MS (HDX-MS), protection factor (PF) analysis, molecular dynamics (MD) simulations, and allostery signaling analysis to examine the structural basis and dynamics for the allosteric regulation of GP by phosphorylation. nTD-MS reveals differences in structural stability as well as oligomeric state between the unphosphorylated (GPb) and phosphorylated (GPa) forms. HDX-MS, PF analysis, and MD simulations further pinpoint the structural differences between GPb and GPa involving the binding interfaces (the N-terminal and tower-tower helices), catalytic site, and PLP-binding region. More importantly, it also allowed us to complete the missing link of the long-range communication process from the N-terminal tail to the catalytic site caused by phosphorylation. This integrative MS and in silico-based platform is highly complementary to biophysical approaches and yields valuable insights into protein structures and dynamic regulation.