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

点击翻译按钮获取中文摘要

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.