Project A01 – 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 Protein X-ray Crystallography & 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 Protein X-ray Crystallography & Signal Transduction
Charitéplatz 1, D-10117 Berlin

Phone +49 30 450 524 178
E-Mail

Nicolas A. Heyder (PhD Student)

Charité - Universitätsmedizin Berlin
Institute for Medical Physics and Biophysics
Group Protein X-ray Crystallography & Signal Transduction
Charitéplatz 1, D-10117 Berlin

Phone +49 30 450 524 170
E-Mail

Further Team Members

Gunnar Kleinau (Senior scientist at Charité)
David Speck (PhD at Charité )
Brian Bauer (Technician at Charité)
Anja Koch (Technician at Charité)

Resources

Methods and Techniques used in A01 and A05 at the Charité:

Structure biology methods:

  • 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 production methods:

  • 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

Biophysical methods:

  • 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

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 PSchöneberg 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

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.

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.

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.

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.

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.

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.

Kim YJ, Hofmann KP, Ernst OP, Scheerer P§, Choe HW§, Sommer ME§. Crystal structure of pre-activated arrestin p44. Nature. 2013; 497:142-146.

Choe HW, Kim YJ, Park JH, Morizumi T, Pai EF, Krauss N, Hofmann KP, Scheerer P, Ernst, OP (2011) Crystal structure of metarhodopsin II. Nature. 471: 651-5.

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.

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.