National Laboratory of Solid State Microstructure and Department of Physics, Nanjing University, Nanjing 210093, China; and.
National Laboratory of Solid State Microstructure and Department of Physics, Nanjing University, Nanjing 210093, China; and
Proc Natl Acad Sci U S A. 2014 Jul 22;111(29):10550-5. doi: 10.1073/pnas.1402768111. Epub 2014 Jul 7.
Ligand binding modulates the energy landscape of proteins, thus altering their folding and allosteric conformational dynamics. To investigate such interplay, calmodulin has been a model protein. Despite much attention, fully resolved mechanisms of calmodulin folding/binding have not been elucidated. Here, by constructing a computational model that can integrate folding, binding, and allosteric motions, we studied in-depth folding of isolated calmodulin domains coupled with binding of two calcium ions and associated allosteric conformational changes. First, mechanically pulled simulations revealed coexistence of three distinct conformational states: the unfolded, the closed, and the open states, which is in accord with and augments structural understanding of recent single-molecule experiments. Second, near the denaturation temperature, we found the same three conformational states as well as three distinct binding states: zero, one, and two calcium ion bound states, leading to as many as nine states. Third, in terms of the nine-state representation, we found multiroute folding/binding pathways and shifts in their probabilities with the calcium concentration. At a lower calcium concentration, "combined spontaneous folding and induced fit" occurs, whereas at a higher concentration, "binding-induced folding" dominates. Even without calcium binding, we observed that the folding pathway of calmodulin domains can be modulated by the presence of metastable states. Finally, full-length calmodulin also exhibited an intriguing coupling between two domains when applying tension.
配体结合调节蛋白质的能量景观，从而改变它们的折叠和变构构象动力学。为了研究这种相互作用，钙调蛋白一直是一种模型蛋白。尽管受到了广泛关注，但钙调蛋白折叠/结合的完全解析机制尚未阐明。在这里，通过构建一个可以整合折叠、结合和变构运动的计算模型，我们深入研究了与两个钙离子结合和相关变构构象变化相关的分离钙调蛋白结构域的折叠。首先，力学拉伸模拟揭示了三种不同构象状态的共存：未折叠状态、闭合状态和开放状态，这与最近的单分子实验的结构理解一致，并增强了这种理解。其次，在接近变性温度时，我们发现了相同的三种构象状态和三种不同的结合状态：零、一和两个钙离子结合状态，导致多达九个状态。第三，根据九态表示，我们发现了折叠/结合的多途径和它们的概率随钙离子浓度的变化。在较低的钙离子浓度下，“自发折叠和诱导契合的结合”发生，而在较高的浓度下，“结合诱导的折叠”占主导地位。即使没有钙离子结合，我们也观察到钙调蛋白结构域的折叠途径可以通过亚稳态的存在来调节。最后，当施加张力时，全长钙调蛋白也表现出两个结构域之间的一种有趣的耦合。
