Project A01 – Structural elucidation of activity regulation at GPCRs involved in metabolism and immune responses
The melanocortin-4 receptor (MC4R) is a highly potent pharmacological target for the treatment of obesity, as it contributes significantly to the regulation of body weight and a high number of receptor variants have been identified in obese or overweight patients. Project A01 aims to determine high-resolution 3D structures of MC4R in yet unknown conformations and complexes associated with specific functional properties and pathogenic conditions. The goal is to gain a more detailed understanding of the impact of internal and external factors on receptor signaling. The project will be focus on complexes between MC4R and various ligands with the G-protein subtype Gq/11, with arrestin and with the interacting transmembrane protein melanocortin-2 receptor accessory protein 2 (MRAP2). In addition, several MC4R variants with pathogenic functional alterations will be structurally studied. To achieve these objectives, advanced membrane protein production technolo-gies, biophysical and cell-based assays, protein X-ray crystallography and cryo-electron microscopy are applied. The gained insights are fundamental for the understanding of activation, selectivity, transient and allosteric processes at this receptor and for the development and optimization of pharmacological tools such as melanocortin derivatives and novel non-peptidic drugs.
Structural elucidation of neuropeptide GPCRs
Project A01 aims to determine high-resolution 3D-structures for the melanocortin-4 receptor (MC4R) and the growth hormone secretagogue receptor (GHSR). Both are neuropeptide class A GPCRs that are of high medical and pharmacological interest. They contribute to the regulation of appetite and metabolism, but also expose structural features which are not common in class A GPCRs. The atomic structural complexes in different signaling states will help to clarify unknown details of the dynamic signal transduction process. For this task advanced technologies in membrane protein production, biophysical assays, protein X-ray crystallography and cryo-electron microscopy will be used.
Contact
Dr. Patrick Scheerer (Project Leader)
Charité - Universitätsmedizin Berlin
Institute for Medical Physics and Biophysics
Group Structural Biology of Cellular Signaling
(Protein X-ray Crystallography - Cryo-Electron Microscopy - Signal Transduction)
Charitéplatz 1, D-10117 Berlin
Phone +49 30 450 524 178
E-Mail
Web biophysik.charite.de/forschung/ag_scheerer
Dr. Michal Szczepek (Scientist)
Charité - Universitätsmedizin Berlin
Institute for Medical Physics and Biophysics
Group Structural Biology of Cellular Signaling
(Protein X-ray Crystallography - Cryo-Electron Microscopy - Signal Transduction)
Charitéplatz 1, D-10117 Berlin
Phone +49 30 450 524 178
E-Mail
Dr. Antje Kamprad (Scientist)
Charité - Universitätsmedizin Berlin
Institute for Medical Physics and Biophysics
Group Structural Biology of Cellular Signaling
(Protein X-ray Crystallography - Cryo-Electron Microscopy - Signal Transduction)
Charitéplatz 1, D-10117 Berlin
Phone +49 30 450 524 170
E-Mail
Monique Gallandi (PhD Student)
Charité - Universitätsmedizin Berlin
Institute for Medical Physics and Biophysics
Group Structural Biology of Cellular Signaling
(Protein X-ray Crystallography - Cryo-Electron Microscopy - Signal Transduction)
Charitéplatz 1, D-10117 Berlin
Phone +49 30 450 524 170
E-Mail
Further Team Members
Dr. Gunnar Kleinau (Senior scientist at Charité)
Anja Koch (Technician at Charité)
Dr. Andrea Schmidt (Senior scientist at Charité)
Fromer Team Member
Brian Bauer (Technician at Charité)
Dr. Nikolas Heyder (PhD Student until 10/2022)
Resources
- Protein X-ray crystallography, membrane protein crystallization, lipidic cubic phase (LCP) crystallization (e.g. TTP Labtech’s mosquito, Gryphon-LCP crystallization and PRIMA Xtallob robots; Formulatrix – Rock Imager 182/54; High-end Stereo microscope Leica M205; Formulatrix – MUVIS)
- X-ray data acquisition: synchrotrons (e.g. BESSY and DESY (Germany), ESRF (France), or in house rotating anode generator (MicroMax007 Microfocus)
- Free-electron laser (XFEL) X-ray data acquisition: (e.g. LCLS-SLAC (USA) or SACLA-Spring-8 (Japan))
- Cryo-electron microscopy (in house) – high-end 300 kV FEI Titan Krios G3 cyro-TEM System/ K3 direct electron detector/ Volta phase plate/ energy filter, Vitrobot, 120kV TEM for sample screening
- GPCR cloning/expression/purification/solubilisation methods
- Large-scale heterologous cell expression (baculovirus expression in Sf9/High Five™ and HEK or Expi293F and E.Coli)
- Various purification systems: Äktapurifier (FPLC), AktaprimePlus (Gel filtration) or ultra-high-performance liquid chromatography – UltiMate 3000 Bio UHPLC
- Production of G-protein or arrestin
- Nanodisc/SMA lipid particles (SMALPs) production and integration
- NanoBRET Ligand-Binding and G-protein Dissociation assays
- cAMP Signaling assays (in vivo)
- Nano-differential scanning fluorimetry (nDSF with NanoTemper Prometheus system) or other thermal shift assays (e.g. CPM) to test thermostability
- MicroScale Thermophoresis (MST) to test binding affinity & protein interactions (NanoTemper Monolith NT 115 system)
- Multiplate reader for assay development with e.g. BRET/FRET/nanoLuc (CLARIOstar Plus – BMG Labtech)
- Surface plasmon resonance-like instrument to test biomolecular interaction (White FOx 1.0 – FOx Biosystem)
- Static light scattering – Multi-angle light scattering detector (Wyatt Technology)
- UV-vis spectrometer (Cary4000, Agilent)
Publications
Bredow C, Thery F, Wirth EK, Ochs S, Kespohl M, Kleinau G, Kelm N, Gimber N, Schmoranzer J, Voss M, Klingel K, Spranger J, Renko K, Ralser M, Mülleder M, Heuser A, Knobeloch KP, Scheerer P, Kirwan J, Brüning U, Berndt N, Impens F, Beling A. ISG15 blocks cardiac glycolysis and ensures sufficient mitochondrial energy production during Coxsackievirus B3 infection. Cardiovasc Res. 2024 Feb 3:cvae026. doi: 10.1093/cvr/cvae026 . Epub ahead of print. PMID: 38309955 .
Makkonen K, Jännäri M, Crisóstomo L, Kuusi M, Patyra K, Melnyk V, Linnossuo V, Ojala J, Ravi R, Löf C, Mäkelä JA, Miettinen P, Laakso S, Ojaniemi M, Jääskeläinen J, Laakso M, Bossowski F, Sawicka B, Stożek K, Bossowski A, Kleinau G, Scheerer P, FinnGen F, Reeve MP, Kero J. Mechanisms of thyrotropin receptor-mediated phenotype variability deciphered by gene mutations and M453T-knockin model. JCI Insight. 2024 Jan 9;9(4):e167092. doi: 10.1172/jci.insight.167092 . PMID: 38194289 .
Sohail I, Laurin SA, Kleinau G, Chunila V , Kagiali ZCUK, Lohse MJ, Scheerer P, Bouvier M, McCormick P, Annibale P, Biebermann H. MRAP2 modifies the signaling and oligomerization state of the melanocortin-4 receptor. bioRxiv 2024.; doi: https://doi.org/10.1101/2024.04.09.588099 .
Kleinau G, Ali AH, Wiechert F, Szczepek M, Schmidt A, Spahn CMT, Liebscher I, Schöneberg T, Scheerer P. Intramolecular activity regulation of adhesion GPCRs in light of recent structural and evolutionary information. Pharmacol Res. 2023 Nov;197:106971. doi: 10.1016/j.phrs.2023.106971. Epub 2023 Oct 30. PMID: 38032292
Giesecke Y, Asimi V, Stulberg V, Kleinau G, Scheerer P, Koksch B, Grötzinger C. Is the Neuropeptide PEN a Ligand of GPR83? Int J Mol Sci. 2023 Oct 12;24(20):15117. doi: 10.3390/ijms242015117. PMID: 37894796 F
Smirnova J, Loerke J, Kleinau G, Schmidt A, Bürger J, Meyer EH, Mielke T, Scheerer P, Bock R, Spahn CMT, Zoschke R. Structure of the actively translating plant 80S ribosome at 2.2 Å resolution. Nat Plants. 2023 Jun;9(6):987-1000. doi: 10.1038/s41477-023-01407-y. Epub 2023 May 8. PMID: 37156858
Hegner B, Kretzschmar T, Zhu N, Kleinau G, Zhao H, Kamhieh-Milz J, Hilger J, Schindler R, Scheerer P, Riemekasten G, Philippe A, Catar R. Autoimmune activation and hypersensitization of the AT1 and ETA receptors contributes to vascular injury in scleroderma renal crisis. Rheumatology (Oxford). 2023 Jun 1;62(6):2284-2293. doi: 10.1093/rheumatology/keac594. PMID: 36227102
Catar R, Herse-Naether M, Zhu N, Wagner P, Wischnewski O, Kusch A, Kamhieh-Milz J, Eisenreich A, Rauch U, Hegner B, Heidecke H, Kill A, Riemekasten G, Kleinau G, Scheerer P, Dragun D, Philippe A. Autoantibodies Targeting AT1- and ETA-Receptors Link Endothelial Proliferation and Coagulation via Ets-1 Transcription Factor. Int J Mol Sci. 2022, 23, 244. doi: 10.3390/ijms23010244. PMID: 35008670.
Höpfner F, Paisdzior S, Reininghaus N, Sohail I, Scheerer P, Annibale P, Biebermann H, Kühnen P. Evaluation of pharmacological rescue of Melanocortin-4 receptor nonsense mutations by aminoglycoside. Life. 2022; 12(11).
Nemec K, Schihada H, Kleinau G, Zabel U, Grushevskyi EO, Scheerer P, Lohse MJ, Maiellaro I. Functional modulation of PTH1R activation and signaling by RAMP2. Proc Natl Acad Sci U S A. 2022 Aug 9;119(32):e2122037119. doi: 10.1073/pnas.2122037119 . Epub 2022 Aug 1. PMID: 35914163 .
Philippe A, Kleinau G, Gruner JJ, Wu S, Postpieszala D, Speck D, Heidecke H, Dowell SJ, Riemekasten G, Hildebrand PW, Kamhieh-Milz J, Catar R, Szczepek M, Dragun D, Scheerer P. Molecular Effects of Auto-Antibodies on Angiotensin II Type 1 Receptor Signaling and Cell Proliferation. Int J Mol Sci. 2022 Apr 2;23(7):3984. doi: 10.3390/ijms23073984. PMID: 35409344
Reininghaus N, Paisdzior S, Höpfner F, Jyrch S, Cetindag C, Scheerer P, Kühnen P, Biebermann H. A Setmelanotide-like Effect at MC4R Is Achieved by MC4R Dimer Separation. Biomolecules. 2022 Aug 15;12(8):1119. doi: 10.3390/biom12081119. PMID: 36009013.
Schulze AS, Kleinau G, Krakowsky R, Rochmann D, Das R, Worth CL, Krumbholz P, Scheerer P, Stäubert C. Evolutionary analyses reveal immune cell receptor GPR84 as a conserved receptor for bacteria-derived molecules. iScience. 2022 Sep 6;25(10):105087. doi: 10.1016/j.isci.2022.105087. eCollection 2022 Oct 21. PMID: 36164652.
Speck D, Kleinau G, Szczepek M, Kwiatkowski D, Catar R, Philippe A, Scheerer P. Angiotensin and Endothelin Receptor Structures With Implications for Signaling Regulation and Pharmacological Targeting. Front Endocrinol (Lausanne). 2022 Apr 19;13:880002. doi: 10.3389/fendo.2022.880002. eCollection 2022. PMID: 35518926.
Speck D, Kleinau G, Meininghaus M, Erbe A, Einfeldt A, Szczepek M, Scheerer P, Pütter V. Expression and Characterization of Relaxin Family Peptide Receptor 1 Variants. Front Pharmacol. 2022 Jan 28;12:826112. doi: 10.3389/fphar.2021.826112. eCollection 2021. PMID: 35153771 Free PMC article.
Voss M, Kleinau G, Gimber N, Janek K, Bredow C, Thery F, Impens F, Schmoranzer J, Scheerer P, Kloetzel PM, Beling A. A cytosolic disulfide bridge-supported dimerization is crucial for stability and cellular distribution of Coxsackievirus B3 protein 3A. FEBS. 2022. https://doi.org/10.1111/febs.16368.
Yang Y, Stensitzki T, Sauthof L, Schmidt A, Piwowarski P, Velazquez Escobar F, Michael N, Nguyen AD, Szczepek M, Brünig FN, Netz RR, Mroginski MA, Adam S, Bartl F, Schapiro I, Hildebrandt P, Scheerer P, Heyne K. Ultrafast proton-coupled isomerization in the phototransformation of phytochrome. Nat Chem. 2022 May 16. doi: 10.1038/s41557-022-00944-x. Online ahead of print. PMID: 35577919.
Chen J, Bi H, Pettersson ME, Sato DX, Fuentes-Pardo AP, Mo C, Younis S, Wallerman O, Jern P, Molés G, Gómez A, Kleinau G, Scheerer P, Andersson L. Functional differences between TSHR alleles associate with variation in spawning season in Atlantic herring. Commun Biol. 2021 Jun 25;4(1):795. doi: 10.1038/s42003-021-02307-7. PMID: 34172814.
Heyder NA, Kleinau G, Speck D, Schmidt A, Paisdzior S, Szczepek M, Bauer B, Koch A, Gallandi M, Kwiatkowski D, Bürger J, Mielke T, Beck-Sickinger A, Hildebrand P, Spahn CMT, Hilger D, Schacherl M, Biebermann H, Hilal T, Kühnen P, Kobilka BK, Scheerer P. Structures of active melanocortin-4 receptor−Gs-protein complexes with NDP-α-MSH and setmelanotide. Cell Research. 2021, 31(11):1176-1189. doi: 10.1038/s41422-021-00569-8. PMID: 34561620.
Hong TI, Hwang KS, Choi TI, Kleinau G, Scheerer P, Bang JK, Jung SH, Kim CH. Zebrafish Bioassay for Screening Therapeutic Candidates Based on Melanotrophic Activity. Int J Mol Sci. 2021; 22:9313. https://doi.org/10.3390/ijms22179313.
Wallach T, Mossmann ZJ, Szczepek M, Wetzel M, Machado R, Raden M, Miladi M, Kleinau G, Krüger C, Dembny P, Adler D, Zhai Y, Kumbol V, Dzaye O, Schüler J, Futschik M, Backofen R, Scheerer P, Lehnardt S. MicroRNA-100-5p and microRNA-298-5p released from apoptotic cortical neurons are endogenous Toll-like receptor 7/8 ligands that contribute to neurodegeneration. Mol Neurodegener. 2021 Nov 27;16(1):80. doi:10.1186/s13024-021-00498-5. PMID: 34838071.
Heyder N, Kleinau G, Szczepek M, Kwiatkowski D, Speck D, Soletto L, Cerdá-Reverter JM, Krude H, Kühnen P, Biebermann H, Scheerer P. Signal Transduction and Pathogenic Modifications at the Melanocortin-4 Receptor: A Structural Perspective. Front Endocrinol (Lausanne). 2019 Jul 31;10:515. doi: 10.3389/fendo.2019.00515. eCollection 2019. PMID: 31417496
Kleinau G, Heyder NA, Tao YX, Scheerer P. Structural Complexity and Plasticity of Signaling Regulation at the Melanocortin-4 Receptor. Int J Mol Sci. 2020 Aug 10;21(16):5728. doi: 10.3390/ijms21165728. PMID: 32785054
Paisdzior S, Dimitriou IM, Schöpe PC, Annibale P, Scheerer P, Krude H, Lohse MJ, Biebermann H, Kühnen P. Differential Signaling Profiles of MC4R Mutations with Three Different Ligands. Int J Mol Sci. 2020 Feb 12;21(4). pii: E1224. doi: 10.3390/ijms21041224. PMID: 32059383
Schulze A, Kleinau G, Neumann S, Scheerer P, Schöneberg T, Brüser A. The intramolecular agonist is obligate for activation of glycoprotein hormone receptors. FASEB J. 2020 Jul 10. doi: 10.1096/fj.202000100R. Epub ahead of print. PMID: 32648604
Behrmann E, Loerke J, Budkevich TV, Yamamoto K, Schmidt A, Penczek PA, Vos MR, Burger J, Mielke T, Scheerer P, Spahn CM. Structural snapshots of actively translating human ribosomes. Cell. 2015; 161:845-57.
Choe HW, Kim YJ, Park JH, Morizumi T, Pai EF, Krauss N, Hofmann KP, Scheerer P, Ernst, OP. Crystal structure of metarhodopsin II. Nature. 2011; 471: 651-5.
Clément K, Biebermann H, Farooqi IS, Van der Ploeg L, Wolters B, Poitou C, Puder L, Fiedorek F, Gottesdiener K, Kleinau G, Heyder N, Scheerer P, Blume-Peytavi U, Jahnke I, Sharma S, Mokrosinski J, Wiegand S, Müller A, Weiß K, Mai K, Spranger J, Grüters A, Blankenstein O, Krude H, Kühnen P. MC4R agonism promotes durable weight loss in patients with leptin receptor deficiency. Nature Medicine. 2018; 24:551-5.
Heyder N, Kleinau G, Szczepek M, Kwiatkowski D, Speck D, Soletto L, Cerdá-Reverter JM, Krude H, Kühnen P, Biebermann H, Scheerer P. Signal Transduction and Pathogenic Modifications at the Melanocortin-4 Receptor: A Structural Perspective. Front Endocrinol (Lausanne). 2019; 10:515.
Kim YJ, Hofmann KP, Ernst OP, Scheerer P§, Choe HW§, Sommer ME§. Crystal structure of pre-activated arrestin p44. Nature. 2013; 497:142-146.
Park JH*, Scheerer P*, Hofmann KP, Choe HW, Ernst OP. Crystal structure of the ligand-free G-protein-coupled receptor opsin. Nature. 2008; 454:183-7.
Qureshi BM, Schmidt A, Behrmann E, Bürger J, Mielke T, Spahn CMT, Heck M, Scheerer P§. Mechanistic insights into the role of prenyl-binding protein PrBP/δ in membrane dissociation of phosphodiesterase 6. Nature Communications. 2018; 9:90.
Saleh N*, Kleinau G*, Heyder N, Clark T, Hildebrand PW§, Scheerer P§. Binding, thermodynamics and selectivity of a non-peptide antagonist to the Melanocortin-4 receptor. Frontiers Pharmacol. 2018; 9:560.
Scheerer P, Park JH, Hildebrand PW, Kim YJ, Krauss N, Choe HW, Hofmann KP, Ernst OP. Crystal structure of opsin in its G-protein-interacting conformation. Nature. 2008; 455:497-502.
Soletto L, Hernández Balfagó S, Rocha A, Scheerer P, Kleinau G, Cerdá-Reverter JM. Melanocortin Receptor Accessory Protein 2-Induced Adrenocorticotropic Hormone Response of Human Melanocortin 4 Receptor. Journal of the Endocrine Society. 2018; 3:314-23.
Szczepek M, Beyrière F, Hofmann KP, Elgeti M, Kazmin R, Rose A, Bartl FJ, von Stetten D, Heck M, Sommer ME, Hildebrand PW, Scheerer P§. Crystal structure of a common GPCR-binding interface for G protein and arrestin. Nature Communications. 2014; 5:4801.
