In-cell NMR technology to study protein interactions

描述(申请人提供)：许多人类疾病的病因涉及由异常翻译后修饰(PTM)引起的多组分蛋白质复合体的结构变化。我们建议开发一种新的基于细胞内核磁共振的技术来绘制伴随着细胞内蛋白质-蛋白质相互作用的结构变化(STINT-核磁共振)，并用它来分析具有生物学意义的分子相互作用。与传统的体外核磁共振和X射线结晶学相比，STINT-核磁共振分析大大减少了获得蛋白质复合体的原子分辨率信息所需的时间。得到的数据定义了原子分辨率下相互作用表面的结构细节。通过顺序表达改变蛋白质-蛋白质相互作用的酶活性，STINT-核磁共振还允许我们执行“细胞内的生物化学”，在那里我们可以监测翻译后修饰的结构后果。这一提议的广泛目标是开发一种新的体内原子拆分技术，以在结构水平上了解翻译后修饰如何调节参与重要生物过程的蛋白质复合体。为了实现这一目标，我们有三个具体的目标：1)发展STINT-核磁共振方法来从结构上表征细菌细胞中的多蛋白质相互作用。作为一个生物系统，我们将使用泛素与未修饰或翻译后修饰的内吞蛋白HRs、STAM2、Eps15之间的相互作用。为此，我们将创建STINT-核磁共振兼容的质粒体，能够在细菌中过度表达内吞蛋白、蛋白激酶和单泛素化机制。我们将使用未修饰和修饰的内吞蛋白的不同组合进行一系列STINT-核磁共振实验，以评估PTM引起的结构变化。我们将用修饰的蛋白质复合体的体外研究来补充我们的细胞内核磁共振实验。2)建立真核细胞的STINT-核磁共振，以测试细胞内结构对蛋白质相互作用的影响。为此，我们将优化泛素和STAM2在酵母细胞中的过度表达，以进行STINT-核磁共振实验。3)扩展STINT-核磁共振，高通量筛选能够干扰萌发所需的病毒蛋白与宿主内吞蛋白之间形成的复合体的小分子。我们将创建能够在细菌中过表达病毒HIV p6结构域、内吞蛋白TSG101和HRS、蛋白激酶MEK1和ERK2以及单素化机制的STINT-核磁共振兼容的质粒构建体。我们将使用未修饰和修饰的蛋白质的不同组合进行一系列STINT-核磁共振实验，以确定与复杂形成相关的结构变化。作为STINT-NMR HTS方法学的积极对照，我们将筛选一个针对TSG101指导的HIV-1萌芽的已知拮抗剂的小型文库。稍后，将使用STINT-核磁共振对p6-TSG101-HRS复合体进行筛选，以确定能够干扰病毒萌发的化合物的可能类别。 公共卫生相关性：许多人类疾病是由异常的翻译后修饰引起的，这反过来又导致蛋白质-蛋白质相互作用的变化。我们开发了一种新的技术，STINT-核磁共振，它允许我们通过使用核磁共振光谱来研究细胞内翻译后修饰蛋白质之间的结构相互作用，并以原子分辨率进行研究。我们将应用这项技术来研究可能导致疾病状态的异常结构和分子过程。我们还将开发这项技术来寻找可能调节这些分子过程的类似药物的小分子。

DESCRIPTION (provided by applicant): The etiology of many human diseases involves structural changes in multi-component protein complexes caused by aberrant posttranslational modifications (PTM). We propose to develop a novel in-cell NMR-based technology for mapping the STructural changes that accompany protein-protein INTeractions in the cell (STINT-NMR) and use it to analyze molecular interactions of biological significance. STINT-NMR analysis greatly reduces the time required to obtain atomic resolution information on protein complexes compared to traditional in vitro NMR and x-ray crystallography. The resulting data define structural details of the interacting surfaces at atomic resolution. By sequentially expressing enzymatic activities that modify protein-protein interactions, STINT-NMR also allows us to perform "biochemistry inside the cell", where we can monitor the structural consequences of post-translational modifications. The broad objective of this proposal is to develop a novel in vivo atomic resolution technique to understand on structural level how posttranslational modifications regulate protein complexes involved in important biological processes. To fulfill this objective we have three specific aims: 1) Develop STINT-NMR methodology to structurally characterize multiprotein interactions in bacterial cells. As a biological system, we will use the interactions between Ubiquitin and unmodified or posttranslationally modified endocytic proteins Hrs, STAM2, Eps15. For this aim we will create STINT-NMR compatible plasmid constructs capable of overexpressing endocytic proteins, protein kinases, and monoubiquitination machinery in bacteria. We will perform series of STINT-NMR experiments using different combinations of unmodified and modified endocytic proteins to assess the structural changes resulting from PTM's. We will supplement our in- cell NMR experiments with in vitro studies of the modified protein complexes. 2) Develop STINT-NMR for eukaryotic cells to test the influence of intracellular structures on protein-protein interactions. For this aim we will optimize overexpression of Ubiquitin and STAM2 in yeast cells to perform STINT-NMR experiments. 3) Extend STINT-NMR for high-throughput screening of small molecules capable of interfering with the complexes formed between viral proteins required for budding and host endocytic proteins. We will create STINT-NMR compatible plasmid constructs capable of overexpressing viral HIV p6 domain, endocytic proteins TSG101 and Hrs, protein kinase MEK1 and ERK2, and monoubiquitination machinery in bacteria. We will perform a series of STINT-NMR experiments using different combinations of unmodified and modified proteins to establish structural changes associated with complex formation. As a positive control of STINT-NMR HTS methodology, we will screen a small library of known antagonists against TSG101-directed HIV-1 budding. Later, a library of small drug-like molecules (NCI Discover set) will be screened against the p6-TSG101-Hrs complexes using STINT-NMR to identify possible classes of the compounds capable of interfering with viral budding. PUBLIC HEALTH RELEVANCE: Many human diseases are caused by aberrant post-translational modifications which, it turn, result in changes in protein-protein interactions. We developed a new technology, STINT-NMR, which allows us to study STructural INTeractions between post- translationally modified proteins inside the cell with atomic resolution by using Nuclear Magnetic Resonance (NMR) spectroscopy. We will apply this technology to study aberrant structures and the molecular processes that may lead to disease states. We will also develop this technology to search for small drug-like molecules that may regulate these molecular processes.