Combining Absolute Quantitative Cross-Linking Mass Spectrometry and Molecular Modeling for Probing PROTAC-Mediated Ternary Complex Structures

摘要 交联质谱(CLMS)在蛋白质3D测定中发挥着越来越重要的作用 结构。通过对交联物(即，通过消化交联物产生的交联肽)的结构阐明 连接的蛋白质或蛋白质复合体)，CLMS可以确定特定功能之间的距离限制 可用于指导蛋白质和蛋白质复合体的结构建模和对接的基团。进一步 交联键的定量分析将使探索蛋白质构象的动态变化成为可能 解决方案，这对传统结构生物学技术来说是一项具有挑战性的任务。然而，绝对的定量分析 由于缺乏标准的交联肽，交联肽的研究非常具有挑战性。为了解决这个问题，我们建议 建立一种新的库仑质量绝对定量交联质谱(AqCLMS) 光谱分析(CMS)是我们实验室最近发展起来的一种绝对定量方法，不需要使用 标准。它是基于交联键的电化学氧化产生电流，并且 用MS测量氧化产率，因此不需要使用任何绝对标准 量化。AqCLMS方法将能够：1)所有交联物之间的定量分析和比较 在一个特定或不同的构象状态，大大增加了关于残基可访问性的信息 以及它们与传统CLMS的距离约束；2)暂态绝对量的计算 在特定生物事件期间形成的蛋白质复合体，这很难使用现有的 技巧。我们预见，我们的aqCLMS方法在探测动态蛋白质构象方面是非常有用的， 这将对结构生物学和药物发现产生重大影响。 在这个项目中，我们建议使用这种aqclms方法来探测和量化构象结构。 针对嵌合体(PROTAC)介导的复合体的蛋白分解，特别是BRD4-PROTAC-CRBN。一把钥匙 PROTAC的步骤是形成诱导的蛋白质复合体，其中降解分子招募E3连接酶 到感兴趣蛋白(POI)，以促进POI的泛素化，最终导致其蛋白酶体 退化。因此，PROTAC的合理设计将需要对TARGET-PROTAC-E3的结构理解 然而，三元络合物仍然是非常有限的。在这个项目中，结合先进的分子 动力学(MD)模拟，aqCLMS将提供对PROTAC的动态性质的深刻见解- 中介的三元结构集合超出了在晶体结构中看到的。

Abstract Cross-linking mass spectrometry (CLMS) is playing an increasingly important role in determining protein 3D structures. By structural elucidation of cross-links (i.e., cross-linked peptides resulting from digestion of cross- linked proteins or protein complexes), CLMS can determine distance constraints between specific functional groups that can be used to guide structural modeling and docking of proteins and protein complexes. Further quantitative analysis of cross-links would allow exploration of dynamic protein conformational changes in solution, a challenging task for traditional structural biology techniques. However, absolute quantitative analysis of cross-links is very challenging, due to lack of standard cross-linked peptides. To tackle this issue, we propose to develop a novel absolute quantitative cross-linking mass spectrometry (aqCLMS) using coulometric mass spectrometry (CMS), a method recently developed in our laboratory for absolute quantitation without using standards. It is based on electrochemical oxidation of cross-links to produce electric current and the measurement of the oxidation yield by MS, therefore eliminating the need of using any standards for absolute quantitation. An aqCLMS method would enable: 1) quantitative analysis and comparison among all cross-links at one particular or different conformational states, greatly increasing information about residue accessibilities and their distance constraints in comparison with traditional CLMS; 2) evaluating absolute quantities of transient protein complexes that form during a particular biological event, which is difficult to measure using existing techniques. We foresee that our aqCLMS method is very informative in probing dynamic protein conformations, which would have high impact in structural biology and drug discovery. In this project, we propose to use this aqCLMS approach to probe and quantify the conformational structures of PROteolysis TArgeting Chimera (PROTAC)-mediated complexes, specifically BRD4-PROTAC-CRBN. A key step in PROTAC is the formation of an induced protein complex where a degrader molecule recruits an E3 ligase to the protein of interest (POI) to facilitate the ubiquitination of the POI, eventually leading to its proteasomal degradation. Thus, rational design of PROTACs will require a structural understanding of target-PROTAC-E3 ternary complexes, which is, however, still very limited. In this project, in combination with advanced molecular dynamics (MD) simulations, aqCLMS would provide deep insights into the dynamic nature of the PROTAC- mediated ternary structure ensemble beyond what is seen in the crystal structures.