Department of Urology, Affiliated Hospital of Weifang Medicinal University, Weifang, Shandong, China.
Department of Infectious Diseases, Affiliated Hospital of Weifang Medicinal University, Weifang, Shandong, China.
J Biomol Struct Dyn. 2020 Apr;38(6):1741-1750. doi: 10.1080/07391102.2019.1615988. Epub 2019 May 16.
The autoinhibition phenomenon has been frequently observed in enzymes and represents an important regulatory strategy to fine-tune enzyme activity. Evolution has exploited this mechanism to reduce enzymatic activity. Glycogen synthase kinase 3β (GSK3β) undergoes autoinhibition via the phosphorylation of Ser9 at the N-terminus of the kinase, which, acting as a pseudosubstrate, occupies the catalytic domain of GSK3β and subsequently blocks primed substrates from having access to the catalytic domain. The detailed structural basis of the autoinhibition mechanism of GSK3β by the pseudosubstrate, however, has not yet been fully resolved. Here, a three-dimensional model of the binary GSK3β-pseudosubstrate complex was built via the molecular modeling method. Based on the constructed model, extensive molecular dynamics (MD) simulations and subsequent molecular mechanics generalized Born/surface area (MM_GBSA) calculations were performed on the wild-type GSK3β-pseudosubstrate complex and three mutated systems (R4A, R6A, and S9A). Analyses of MD simulations and binding free energies revealed that the phosphorylation of Ser9 is the prerequisite for the autoinhibition of GSK3β, and both mutations of Arg4 and Arg6 to alanine markedly reduced the binding affinities of the mutated pseudosubstrate to the GSK3β catalytic domain, thereby disrupting the autoinhibition of the kinase. This study highlights the importance of Ser9, Arg6, and Arg4 in modulating the autoinhibition mechanism of GSK3β, contributing to a deeper understanding of GSK3β biology.Communicated by Ramaswamy H. Sarma.
自动抑制现象在酶中经常被观察到，是一种微调酶活性的重要调控策略。进化利用这种机制来降低酶的活性。糖原合酶激酶 3β（GSK3β）通过其激酶 N 端丝氨酸 9 的磷酸化而发生自动抑制，该丝氨酸 9 作为伪底物，占据 GSK3β 的催化结构域，并随后阻止已磷酸化的底物进入催化结构域。然而，GSK3β 伪底物的自动抑制机制的详细结构基础尚未完全阐明。在这里，通过分子建模方法构建了二元 GSK3β-伪底物复合物的三维模型。基于构建的模型，对野生型 GSK3β-伪底物复合物和三个突变系统（R4A、R6A 和 S9A）进行了广泛的分子动力学（MD）模拟和随后的分子力学广义 Born/表面积（MM_GBSA）计算。MD 模拟和结合自由能分析表明，Ser9 的磷酸化是 GSK3β 自动抑制的前提条件，Arg4 和 Arg6 突变为丙氨酸显著降低了突变伪底物与 GSK3β 催化结构域的结合亲和力，从而破坏了激酶的自动抑制。这项研究强调了 Ser9、Arg6 和 Arg4 在调节 GSK3β 自动抑制机制中的重要性，有助于深入了解 GSK3β 生物学。
