OPENING ION CHANNELS WITH A TRANS-CIS PROLINE SWITCH: A COMPUTATIONAL METADYNAMICS STUDY

配体门控离子通道(LGICs)是神经元传递的重要介体，与阿尔茨海默病、帕金森病等多种神经系统疾病密切相关。这些位于神经细胞膜上的受体是由离子传导中心孔周围排列的不同亚基组成的大蛋白，每个亚基由一个胞外区、一个跨膜区和一个胞内区组成。LGICs的激活是由一个小的神经递质与细胞外结构域结合启动的：这触发了一系列化学事件和蛋白质的构象变化，最终导致通道的开放(门控)：然后离子可以涌入细胞膜，改变细胞的活动。关于LGICs最耐人寻味的问题是，细胞外域的一个小神经递质的结合如何转化为超过50+远的跨膜域的通道开放。最近关于5-羟色胺3型受体(5-HT3R)的实验表明，位于两个跨膜螺旋(M2-M3环)之间的环(M2-M3环)顶端的一个特定的脯氨酸氨基酸(Pro 8*)可以通过跨顺式异构化及其对蛋白质骨架的结构效应来作为离子通道门控的开关。由于LGIC的结构信息有限，原子模拟可以在验证实验支持的门控机制方面发挥关键作用，提供更多的见解。特别是，这个探索性计算项目的目标是证明元动力学(一种探索复杂多原子系统自由能表面的新模拟技术)的使用是否可以提供所提议的门控机制的原子学图像。我们将结合经典和量子力学的方法，开始研究一个模拟5-HT3R相关(M2-M3)环的20个氨基酸的模型肽中的脯氨酸开关。我们将评估在实验中产生功能性或非功能性受体的顺式或反式构象，以及环境对异构化机制的影响。然后，我们将增加模型系统的复杂性，例如，包括被认为由Pro开关重新定位的通道衬里(M2)螺旋。最后，我们将把分析扩展到与5-HT3R相同的超家族的受体，寻找相似或替代的开关。这项拟议的研究将导致开发使用元动力学技术来研究生物分子中的开关机制的协议。

Ligand-Gated ion Channels (LGICs) are important mediators in neuronal transmission and are involved in many neurological disorders, such as Alzheimer's and Parkinson's diseases. These receptors, located in the membrane of nerve cells, are large proteins, consisting of different subunits arranged around an ion-conducting central pore; each subunit is composed by an extracellular domain, a transmembrane domain and an intracellular domain. The activation of LGICs is initiated by the binding of a small neurotransmitter to the extracellular domain: this triggers a series of chemical events and conformational changes in the protein culminating with the opening (gating) of the channel: ions can then flood across the cell membrane modifying the cell activity. The most intriguing question concerning LGICs is how the binding of a small neurotransmitter in the extracellular domain translates into the opening of the channel in the transmembrane domain more than 50 + away. Recent experiments on the 5-hydroxytryptamine type 3 receptor (5-HT3R) led to the proposal that a specific proline amino-acid (Pro 8*), located at the apex of a loop between two transmembrane helices (M2-M3 loop), can act as a switch for ion channel gating by means of a trans-cis isomerisation and of its structural effects on the protein backbone. Since structural information on LGICs is limited, atomistic simulations can play a crucial role in verifying the gating mechanism supported by the experiments, providing additional insights. In particular, the goal of this exploratory computational project is to demonstrate whether the use of metadynamics (a novel simulation technique to explore the free energy surfaces of complex polyatomic systems) can provide an atomistic picture of the proposed gating mechanism. Using a combination of classical and quantum mechanical methods, we will start investigating the proline switch in a 20 amino-acid model peptide mimicking the 5-HT3R relevant (M2-M3) loop. We will assess the effects of proline mutations with proline analogues, preferring either the cis or the trans conformations, which in experiments produced functional or non-functional receptors, and evaluate the influence of the environment on the isomerisation mechanism. We will then increase the complexity of the model system, e.g. including the channel lining (M2) helix which is thought to be repositioned by the proline switch. Finally, we will extend the analysis to receptors of the same superfamily as 5-HT3R, searching for similar or alternative switches. The proposed research will lead to the development of protocols for the use of the metadynamics technique to study switching mechanisms in biomolecules.