Integrating HDX-MS and Molecular Dynamics to investigate the mechanism of activation of the RORy by multiple small molecule modulators

关于激活机制的详细信息对许多蛋白质来说是个谜。蛋白质的机制和功能是系统结构和动力学的产物。对于X射线结晶学和低温电子显微镜等标准结构研究来说，动力学行为是不可见的。为了提高我们对蛋白质药物靶标如何对配体作出反应的理解，需要开发能够探测动力学的方法。氢-氚交换质谱仪(HDX-MS)是一种广泛使用的实验技术，它提供了关于蛋白质的酰胺氢键网络的定量信息，从而促进了对蛋白质构象动力学的探索。HDX-MS在其他补充生物物理技术中脱颖而出，因为它比许多其他方法有优势，因为它可以研究的蛋白质大小没有限制，而且它可以在类似于自然环境的条件下探测蛋白质。因此，HDX-MS被广泛用于小分子、抗体和疫苗应用。在小分子研究中，HDX-MS可以报告直接结合事件和变构调节。在构象机理研究中，通过限制蛋白质的构象状态，可以绕过从HDX数据到结构表示的完全反卷积。这类实验研究往往得到分子动力学(MD)模拟的补充，分子动力学模拟可用于监测蛋白质及其复合体的构象动力学。HDX-MS在抗体治疗研究中的频繁使用突出了其作为分析科学常规使用分支的地位。该实验方法结合了生物信息学和计算与理论化学。HDX-MS涉及将蛋白质分解成多肽，然后必须匹配多肽片段/蛋白质序列，这可能是具有挑战性的，因为有许多不同的多肽/质量可能性。因此，MD模拟被用来帮助解决结果数据中的差距，并帮助将空间分布的汇率与结构函数联系起来。由于蛋白质本质上是非线性系统，MD模拟需要仔细实施，以将动态观测重新加权到模拟结构，将动力学与蛋白质功能联系起来。目前，进行HDX-MS研究主要有两种方式：丰富的实验和丰富的模拟。主要的研究目标是比较这两种方法，并应用HDX-MS和MD模拟的最新技术。这一目标将通过该团队的跨学科经验的结合来实现：实验HDX-MS(斯里纳特·克里希纳穆尔蒂，OMassy)，计算化学(Maria Musgaard，OMassa)，HDX-MS理论方法(Oliver Crook，牛津)，以及结构生物学和统计学(Charlotte Deane，牛津)。在这个项目中，我们将开发一种HDX-MS和MD相结合的方法来研究多个小分子调节剂激活受体的机制。除了简单的对比最佳实践，我们计划探索的一个有趣的领域是这些数据的图形和可视化，固有的高维度可能会使解释具有挑战性。从已发表的研究中获得了大量的HDX-MS数据，并为比较不同的方法提供了一个试验台。此外，OMasss目前有几项研究正在进行探索，并有可能用进一步的实验来补充数据。这将使我们能够研究HDX-MS实验的最新进展。首先，我们将利用HDX-MS(通过HDX-MS研究38种化合物和大约60个PDB结构)在先前的Rory调节剂结构动力学库中产生的丰富数据，来确定是否可以从试图揭示激活构象动力学的MD轨迹中预测或选择正确的蛋白质-配体复合体构象。

Detailed information on the mechanism of activation is enigmatic for many proteins. Protein mechanism and function is a product of both the structure and the dynamics of the system. Dynamical behaviour is invisible to standard structural studies such as X-ray crystallography and CryoEM. To improve our understanding of how protein drug targets respond to ligands requirements the development of methods that can probe dynamics. Hydrogen-Deuterium Exchange Mass-Spectrometry (HDX-MS) is a widely used experimental technique that provides quantitative information on the protein's amide hydrogen bonding network, thereby facilitating the exploration of protein conformational dynamics. HDX-MS stands out to other complementary biophysical techniques as it has advantages over many other methods in that there is no limit to the size of protein it can study and it can probe proteins in conditions similar to the native environment. As such HDX-MS is extensively used in small molecule, antibody, and vaccine applications. In small molecule research, HDX-MS can report on both direct binding events and allosteric modulation. In studies of conformational mechanism, the full deconvolution of HDX data into structural representation can be bypassed by restricting the conformational state of proteins. These sorts of experimental studies are often complemented by Molecular Dynamics (MD) simulations that can be used to monitor the conformational dynamics of proteins and their complexes. The frequent use of HDX-MS in antibody therapeutics research underscores its position as a routinely utilized branch of Analytical Science. The experimental method combines Biological Informatics and Computational and Theoretical Chemistry. HDX-MS involves breaking down a protein into peptides, the peptide fragment/protein sequences must then be matched which can be challenging due to the number of different peptide/mass possibilities. MD simulations are therefore used to help resolve gaps in the resulting data and to help relate the spatially distributed exchange rates to structural functions. As proteins are inherently Non-Linear Systems, the MD simulations require careful implementation to reweight the dynamical observations to simulated structures, to link dynamics to protein function. Presently, two main regimes exist for performing HDX-MS studies: experiment rich and simulation rich. A primary research objective is to compare the two approaches and apply the latest techniques from both HDX-MS as well as MD simulation. This goal will be achieved by the combination interdisciplinary experience of the team: experimental HDX-MS (Srinath Krishnamurthy, OMass), computational chemistry (Maria Musgaard, OMass), theoretical methods in HDX-MS (Oliver Crook, Oxford) and structural biology and statistics (Charlotte Deane, Oxford). In this project, we will develop an integrative approach between HDX-MS and MD to investigate the mechanism of activation of receptors with multiple small molecule modulators. Beyond simply contrasting best practices, one intriguing area we plan to explore is the Graphics and Visualisation of these data, the inherent high dimensionality can make interpretation challenging. A wealth of HDX-MS data from published studies are available and provide a testbed to compare different approaches. In addition, OMass has several current studies for exploration as well as the possibility to supplement the data with further experiments. This will enable us to investigate the latest advancements in HDX-MS experiments. Initially we will leverage the wealth of data generated in previous work on a library of structural dynamics from RORy modulators by HDX-MS (38 compounds studied via HDX-MS and around 60 pdb structures) to determine whether the correct protein-ligand complex conformations can be predicted or selected from MD trajectories attempting to reveal the conformational dynamics of activation.