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.通过交联的结构解析（即，交联肽是由交联肽的消化产生的， 连接的蛋白质或蛋白质复合物），CLMS可以确定特定功能之间的距离约束 可用于指导蛋白质和蛋白质复合物的结构建模和对接的基团。进一步 交联的定量分析将允许探索蛋白质构象的动态变化， 这是传统结构生物学技术面临的一个挑战。然而，绝对定量分析 由于缺乏标准的交联肽，因此交联的方法非常具有挑战性。为了解决这个问题，我们建议 建立了一种新的绝对定量交联质谱法（aqCLMS）， 质谱法（CMS），最近在我们的实验室开发的方法，用于绝对定量，而不使用 标准它基于交联的电化学氧化以产生电流， 通过MS测量氧化产率，因此无需使用任何绝对标准品 定量一个aqCLMS方法将能够：1）定量分析和比较所有的交联 在一个特定的或不同的构象状态，大大增加了有关残留物的信息， 与传统的CLMS相比， 在特定生物事件期间形成的蛋白质复合物，这是难以使用现有的方法测量的。 技术.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 PROTAC介导的复合物，特别是BRD 4-PROTAC-CRBN。一个关键 PROTAC中的一个步骤是诱导蛋白复合物的形成，其中降解分子募集E3连接酶 目的蛋白（POI），以促进POI的泛素化，最终导致其蛋白酶体 降解因此，PROTAC的合理设计将需要对目标PROTAC-E3的结构了解 三元复合物，这是，然而，仍然非常有限。在这个项目中，结合先进的分子生物学技术， 动力学（MD）模拟，aqCLMS将提供深入了解PROTAC的动态性质， 介导的三元结构系综超出了在晶体结构中所看到的。

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.