Project C05 – Structural dynamics of allosteric coupling in GPCRs
GPCR activation by agonist binding involves a process called ‘allosteric coupling’, i.e. structural changes in the extracellular ligand binding pocket are conformationally linked to the intracellular G protein-binding site. This is a reciprocal process as agonist binding leads to structural changes in the G protein-binding site and G protein-binding changes the conformation of the ligand binding pocket. In addition to G proteins, GPCRs can also signal through b-arrestin. Biased agonists are special agonists which favor one or more signaling pathways over others and may prove therapeutically beneficial. There is a growing body of evidence suggesting that some triggers for ligand bias are located in the extracellular domains of GPCRs. We have shown that some muscarinic agonists impair the closure of the ligand binding pocket and thereby hamper allosteric coupling to the intracellular binding site. These agonists are biased towards G protein-signaling and fail to recruit b-arrestin. Recently, the same mechanism has been demonstrated for dopamine D2, D3, serotonin 5-HT7 and b2-adrenergic receptors.
In this project we aim to study the structural dynamics of allosteric coupling during GPCR activation in intact cells. We will focus on the muscarinic M2 receptor, for which both inactive and active crystal structures are available, indicating the largest conformational changes in the ligand binding pocket upon receptor activation reported so far. By combining unnatural amino acid technology and biorthogonal labeling, we will install pairs of small organic fluorophores at specific sites on the receptor, according to procedures pioneered by us and well established in our lab. We will develop novel extracellular GPCR sensors based on Foerster resonance energy transfer (FRET) that allow monitoring reciprocal movements of single receptor helices and loops with high temporal and spatial resolution. By comparing the FRET signals upon stimulation with an entire toolbox of M2 receptor agonists of varying efficacy including a series of biased agonists, we aim at linking conformational changes of the ligand binding pocket with intracellular signaling profiles. Overall, our study will provide unprecedented structural insights into how to trigger specific intracellular signals from the extracellular side. We expect that our data will have an important impact for rational design of biased agonists with desired signaling profiles.
Dr. Andreas Bock (Project Leader)
Max Delbrück Center for Molecular Medicine
Unit: Signaling processes of receptors
Robert-Rössle-Strasse 10, D-13125 Berlin
Phone +49 30 9406 1745
Prof. Dr. Irene Coin (Project Leader)
Leipzig University, Faculty of Life Sciences
Institute of Biochemistry
Brüderstrasse 34, D-04103 Leipzig
Phone +49 341 97 36980
- Fluorescence microscopy
- FRET / BRET
- High-throughput screening
- Nucleic acid and protein assays
Bock A, Bermudez M. Allosteric coupling and biased agonism in G protein-coupled receptors. FEBS J. 2021 Feb 23. doi: 10.1111/febs.15783. Epub ahead of print. PMID: 33621418.
Işbilir A, Serfling R, Möller J, Thomas R, De Faveri C, Zabel U, Scarselli M, Beck-Sickinger AG, Bock A, Coin I, Lohse MJ, Annibale P. Determination of G-protein–coupled receptor oligomerization by molecular brightness analyses in single cells. Nat Protoc. 2021. https://doi.org/10.1038/s41596-020-00458-1.
Holze J, Bermudez M, Pfeil EM, Kauk M, Bödefeld T, Irmen M, Matera C, Dallanoce C, De Amici M, Holzgrabe U, König GM, Tränkle C, Wolber G, Schrage R, Mohr K, Hoffmann C, Kostenis E, Bock A. Ligand-Specific Allosteric Coupling Controls G-Protein-Coupled Receptor Signaling. ACS Pharmacol Transl Sci. September 2, 2020 DOI: 10.1021/acsptsci.0c00069
Bock A, Annibale P, Konrad C, Hannawacker A, Anton SE, Maiellaro I, Zabel U, Sivaramakrishnan S, Falcke M, Lohse MJ. Optical Mapping of cAMP Signaling at the Nanometer Scale. Cell. 2020 Aug 20:S0092-8674(20)30943-0. doi: 10.1016/j.cell.2020.07.035. Epub ahead of print. PMID: 32846156.
Bock A, Merten N, Schrage R, Dallanoce C, Bätz J, Klöckner J, Schmitz J, Matera C, Simon K, Kebig A, Peters L, Müller A, Schrobang-Ley J, Tränkle C, Hoffmann C, De Amici M, Holzgrabe U, Kostenis E, Mohr K. The allosteric vestibule of a seven transmembrane helical receptor controls G-protein coupling. Nat Commun. 2012; 3:1044.
Bock A, Schrage R, Mohr K. Allosteric modulators targeting CNS muscarinic receptors. Neuropharmacology. 2018; 136:427-37.
Bock A, Chirinda B, Krebs F, Messerer R, Bätz J, Muth M, Dallanoce C, Klingenthal D, Tränkle C, Hoffmann C, De Amici M, Holzgrabe U, Kostenis E, Mohr K. Dynamic ligand binding dictates partial agonism at a G protein-coupled receptor. Nat Chem Biol. 2014; 10:18-20.
Bock A, Bermudez M, Krebs F, Matera C, Chirinda B, Sydow D, Dallanoce C, Holzgrabe U, De Amici M, Lohse MJ, Wolber G, Mohr K. Ligand binding ensembles determine graded agonist efficacies at a G protein-coupled receptor. J Biol Chem. 2016; 291:16375-89.
Bermudez M, Bock A, Krebs F, Holzgrabe U, Mohr K, Lohse MJ, Wolber G. Ligand-specific restriction of extracellular conformational dynamics constrains signaling of the M2 muscarinic receptor. ACS Chem Biol. 2017; 12:1743-8.
Serfling R, Lorenz C, Etzel M, Schicht G, Böttke T, Mörl M, Coin I. Designer tRNAs for efficient incorporation of non-canonical amino acids by the pyrrolysine system in mammalian cells. Selected as breakthrough paper, journal cover. Nucleic Acids Res. 2018; 46:1-10.
Coin I, Perrin MH, Vale WW, Wang L. Photocrosslinkers incorporated into G-Protein-Coupled-Receptors in mammalian cells: a ligand comparison. Selected as Very-Important-Paper (VIP) by referees. Angew Chem Int Ed Engl. 2011; 50:8077-81.
Serfling R, Seidel L, Böttke T, Coin I. Optimizing the genetic incorporation of chemical probes into GPCRs for photo-crosslinking mapping and bioorthogonal chemistry in live mammalian cells. J Vis Exp. 2018; 134.