Figure 1. Encoding unnatural amino acids (Uaas) in living cells. The orthogonal synthetase aminoacylates the orthogonal tRNA with the desired Uaa, and the acylated tRNA incorporates the Uaa in response to a unique codon (usually an amber stop codon).

All functions of living organisms are regulated by sophisticated signaling networks that involve a multitude of interactions between different proteins. Understanding the topology of protein-protein complexes provides important clues for the development of molecules able to interfere with protein interactions, allowing us to modulate signaling pathways for therapeutic purposes. Protein complexes extracted from the cell and reconstituted in appropriate artificial environments can be characterized at high resolution by NMR and crystallography techniques. Alternative methods are needed both to gain information from the natural context of the live cell and to investigate protein targets that elude direct structural characterization.We focus on mapping protein-protein interaction surfaces in the live cell by combining chemical tools and modern molecular biology techniques. In particular, we use the expanded genetic code technology to incorporate crosslinking amino acids into proteins at specific sites. The unnatural moiety is built into the protein bait directly in the live cell through regular ribosomal synthesis, without the need of any additional step in vitro. The crosslinker captures interacting protein partners that come within its range of proximity. Systematic incorporation of the probe throughout interaction domains provides panoramic information about the topology of the interaction surfaces. 

Figure 2. Mapping binding sites on GPCRs. Crosslinking probes genetically incorporated into the receptor capture natural polypeptide ligands in live mammalian cells.

Using this approach, the molecular basis of the interaction between a GPCR and its natural polypeptide ligands has been discovered in live mammalian cells. A photo-crosslinking amino acid has been incorporated at different positions into the corticotropin releasing factor receptor type 1 (CRF1R), a class B GPCR that regulates the response of the organism to stress stimuli and for which no full-length 3D structure is available. CRF1R mutants containing the crosslinker were able to capture different peptide ligands in a selective and site-specific manner (Coin I et al., 2011, Angew. Chem. Int. Ed. Engl., 50, 8077). Systematic photo-crosslinking scanning of the extracellular side of the receptor unveiled the complete shape of the binding pocket for a natural peptide agonist in the transmembrane region (Coin I et al., 2013, Cell, in press). Subsequently, by genetically incorporating a novel chemical crosslinking probe along the pocket, the relative position and orientation of the ligand were uncovered (Xiang Z, Ren H, Hu YS, Coin I et al., 2013, Nature Methods, 10, 885). Overall, the experimental strategy revealed unique structural features of the full-length post-translationally modified GPCR embedded in the native membrane of the live cell, thus providing a key complement to structural data derived from biophysical reductionist approaches (Coin I et al., 2013, Cell, in press).

Projects in the lab are aimed both at understanding at the molecular level how distinct activities of CRF1R and other GPCRs are regulated by different ligands, and at investigating docking domains of classic downstream effectors such as G-proteins and arrestins as well as receptor-associated proteins that regulate receptor functions.

last modified: 16.10.2019


Prof. Dr. Irene Coin
Institute of Biochemistry
Brüderstraße 34
D-04103 Leipzig

Phone: +49 341 97-36980
Fax: +49 341 97-36909