Mapping of protein-peptide and protein-protein interactions by means of genetically encoded photocrosslinkers

该项目旨在建立一个通用的方法来研究肽-蛋白质和蛋白质-蛋白质的相互作用，在完整的哺乳动物细胞的基础上使用非天然氨基酸诱变和光亲和交联。这将为绘制相互作用位点的拓扑结构以及寻找蛋白质之间的未知相互作用提供工具。我们的主要分子靶点是一种膜蛋白，B类GPCR促肾上腺皮质激素释放因子受体1型（CRFR 1），它代表了生物体对应激刺激反应的关键要素。我们将使用CRFR 1突变体的文库，其在所选结构域的每个位置处具有单个可光活化氨基酸p-叠氮基-Phe（Azi），以测试每个位点捕获肽配体的能力。以这种方式，我们最近定位了天然多肽激动剂Ucn-I的CRFR 1的J结构域结合口袋。在项目A中，我们将建立肽配体的交联图，显示不同的Gs/Gi激活模式。同时，我们将研究当受体与特定的G蛋白预偶联时，CRFR 1-Ucn-I复合物内的相互作用位点如何变化。这将首次提供有关触发不同信号通路的分子决定簇的信息。项目B的目的是在单氨基酸水平上表征Pray分子中的交联位点。首先，我们将使用叠氮化物-炔点击反应来实现突变体受体中的Azi残基与在配体的不同位置处掺入的炔丙基甘氨酸残基之间的位点特异性化学交联。同时，我们将开发一种方法来纯化交联受体的基础上使用的标志标签配对的生物素标签。后者将通过分选酶A催化的转肽反应后序引入。酶消化的优化步骤将提供低分子量的交联片段以用MS/MS进行分析。这将允许确定结合口袋中的配体取向并建立第一个详细的结合模型。在项目C中，我们将把带有二苯甲酮和二氮丙啶光交联剂的氨基酸掺入CRFR 1跨膜结构域的脂质暴露位置，以研究参与形成同源二聚体的表面。相同的受体突变体将作为诱饵捕获跨膜区域内相互作用的其他蛋白质，这是目前任何其他可用方法都无法完成的任务。将采用项目B中开发的策略富集交联产物，并采用质谱法鉴定相关蛋白质。同样的方法将用于绘制2型CRFR与癌蛋白ErbB 2的相互作用，并在长期内研究发生在细胞内结构域的其他相互作用。从长远来看，该方法将允许研究在活细胞的天然环境中参与信号级联和其他细胞过程的任何蛋白质-蛋白质相互作用。

The project aims at establishing a general methodology to investigate peptide-protein and protein- protein interactions in intact mammalian cells based on the use of unnatural amino acid mutagenesis and photoaffinity crosslinking. This will provide a tool for mapping the topology of interaction sites as well as for searching for unknown interactions between proteins. Our primary molecular target is a membrane protein, the class-B GPCR c orticotropin releasing factor receptor type 1 (CRFR1), which represents the key element of the organism response to stress stimuli. We will use libraries of CRFR1 mutants bearing a single photoactivatable amino acid, p-azido-Phe (Azi), at each position of selected domains to test the ability of each site to capture peptide ligands. In this way we have very recently located the J-domain binding pocket of CRFR1 for the native polypeptide agonist Ucn-I. In project A we will build crosslinking maps of peptide ligands showing different patterns of Gs/Gi activation. In parallel, we will study how interaction sites within the CRFR1-Ucn-I complex change, when the receptor is pre-coupled to specific G-proteins. This will give first information about molecular determinants that trigger different signaling pathways. Project B aims at characterizing the site of crosslinking in the pray molecule at the single amino acids level. First, we will use the azide-alkyne click reaction to achieve site specific chemical crosslinking between Azi residues in the mutant receptors and propargylglycine residues incorporated at different positions of the ligand. In parallel, we will develop a method to purify crosslinked receptors based on the use of a Flag tag paired to a Biotin tag. The latter will be introduced posttranslationally through a transpeptidation reaction catalyzed by Sortase A. Optimized steps of enzymatic digestion will provide crosslinked fragments of low molecular weight to be analyzed with MS/MS. This will allow determining the ligand orientation in the binding pocket and build the first detailed binding model. In project C we will incorporate amino acids bearing benzophenone and diazirine photocrosslinkers into lipid-exposed positions of the transmembrane domains of CRFR1, to investigate the surfaces involved in the formation of homo-dimers. The same receptor mutants will serve as baits to capture other proteins interacting within the transmembrane region, a task that cannot be accomplished with any other currently available method. Crosslinked products will be enriched with the strategy developed in project B and associated proteins will be identified with mass spectrometry. The same approach will be used to map the interaction of type 2 CRFR with the oncoprotein ErbB2, and in the long term to investigate other interactions taking place at the intracellular domains. In perspective the methodology will allow investigating any protein-protein interaction involved in signaling cascades and other cellular processes in the native environment of the live cell.