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

结合绝对定量交联质谱和分子建模来探测 PROTAC 介导的三元复杂结构

• 批准号: 10572720
• 负责人: Hao Chen
• 金额: $ 24万
• 依托单位: NEW JERSEY INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10572720
• 关键词: Acids; Affinity Chromatography; Amines; Azides; Binding; Biological; Catechols; Chemicals; Complex; Coupled; Cross-Linking Reagents; Data; Development; Digestion; Dimerization; Docking; Equilibrium; Esters; Event; Free Energy; Laboratories; Lysine; Mass Spectrum Analysis; Measurement; Measures; Mediating; Methodology; Methods; Modeling; Molecular; Molecular Conformation; N-terminal; Nature; Outcome; Peptides; Play; Probability; Property; Protac; Protein Conformation; Protein Dynamics; Proteins; Proteomics; Reaction; Research; Resolution; Role; Structural Models; Structure; Sulfoxide; Techniques; Ubiquitination; Validation; crosslink; drug discovery; functional group; improved; insight; interest; molecular dynamics; molecular modeling; novel; oxidation; protein complex; protein crosslink; protein degradation; rational design; recruit; simulation; structural biology; three dimensional structure; ubiquitin-protein ligase

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.

抽象的 交联质谱 (CLMS) 在确定蛋白质 3D 方面发挥着越来越重要的作用 结构。通过交联的结构阐明（即，由交联消化产生的交联肽） 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限制 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。更远 交联的定量分析将允许探索动态蛋白质构象变化 解决方案，对于传统结构生物学技术来说是一项具有挑战性的任务。然而，绝对定量分析 由于缺乏标准的交联肽，交联的建立非常具有挑战性。为了解决这个问题，我们建议 开发一种使用库仑质量的新型绝对定量交联质谱法 (aqCLMS) 光谱法 (CMS)，我们实验室最近开发的一种方法，无需使用 标准。它基于交联的电化学氧化来产生电流和 通过 MS 测量氧化产率，因此无需使用任何绝对标准 定量。 aqCLMS 方法将能够：1）所有交叉链接之间的定量分析和比较 在一种特定或不同的构象状态下，大大增加了有关残基可及性的信息 以及与传统 CLMS 相比的距离限制； 2) 评估瞬态的绝对量 在特定生物事件期间形成的蛋白质复合物，使用现有技术很难测量 技术。我们预见我们的 aqCLMS 方法在探测动态蛋白质构象方面信息丰富， 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 蛋白水解靶向嵌合体 (PROTAC) 介导的复合物，特别是 BRD4-PROTAC-CRBN。一把钥匙 PROTAC 中的一步是诱导蛋白复合物的形成，其中降解分子招募 E3 连接酶 到感兴趣的蛋白质（POI），以促进 POI 的泛素化，最终导致其蛋白酶体 降解。因此，PROTAC 的合理设计需要对目标 PROTAC-E3 有结构性的了解 三元配合物，但仍然非常有限。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- 动态性质的深入见解 介导的三元结构系综超出了晶体结构中所见的范围。