Conformational Changes in Protein Loops and Helices Induced by Post-Translational Phosphorylation

Post-translational phosphorylation is a ubiquitous mechanism for modulating protein activity and protein-protein interactions. In this work, we examine how phosphorylation can modulate the conformation of a protein by changing the energy landscape. We present a molecular mechanics method in which we phosphorylate proteins in silico and then predict how the conformation of the protein will change in response to phosphorylation. We apply this method to a test set comprised of proteins with both phosphorylated and non-phosphorylated crystal structures, and demonstrate that it is possible to predict localized phosphorylation-induced conformational changes, or the absence of conformational changes, with near-atomic accuracy in most cases. Examples of proteins used for testing our methods include kinases and prokaryotic response regulators. Through a detailed case study of cyclin-dependent kinase 2, we also illustrate how the computational methods can be used to provide new understanding of how phosphorylation drives conformational change, why substituting Glu or Asp for a phosphorylated amino acid does not always mimic the effects of phosphorylation, and how a phosphatase can “capture” a phosphorylated amino acid. This work illustrates how computational methods can be used to elucidate principles and mechanisms of post-translational phosphorylation, which can ultimately help to bridge the gap between the number of known sites of phosphorylation and the number of structures of phosphorylated proteins.

翻译后磷酸化（post-translational phosphorylation）是一类广泛存在的调控蛋白质活性与蛋白质间相互作用的机制。本研究探讨了磷酸化如何通过改变能量景观来调控蛋白质构象。我们提出一种分子力学方法，通过计算机模拟对蛋白质进行磷酸化修饰，并预测蛋白质构象响应磷酸化修饰的变化情况。我们将该方法应用于一套同时包含磷酸化与非磷酸化晶体结构的蛋白质测试集，结果证明在大多数情况下，本方法能够以近原子级精度预测磷酸化诱导的局部构象变化，或无构象变化的情况。用于测试该方法的蛋白质样本包括激酶（kinases）与原核生物应答调节蛋白（prokaryotic response regulators）。通过对细胞周期蛋白依赖性激酶2（cyclin-dependent kinase 2）的详细案例分析，本研究还阐释了计算方法如何为理解磷酸化驱动构象变化的机制、为何用谷氨酸（Glu）或天冬氨酸（Asp）替代磷酸化氨基酸并不能总是模拟磷酸化的效应，以及磷酸酶如何“捕获”磷酸化氨基酸提供全新认知。本研究展示了计算方法可用于阐明翻译后磷酸化的相关原理与机制，最终有助于缩小已知磷酸化位点数量与磷酸化蛋白质结构数量之间的差距。