Probing the Conformational Space of the Cannabinoid Receptor 2 and a Systematic Investigation of DNP-Enhanced MAS NMR Spectroscopy of Proteins in Detergent Micelles

Overview

Journal ACS Omega

Publisher American Chemical Society

Specialty Chemistry

Date 2023 Sep 18

PMID 37720784

Authors

Johanna Becker-Baldus

Alexei Yeliseev

Thomas T Joseph

Snorri Th Sigurdsson

Lioudmila Zoubak

Kirk Hines

Malliga R Iyer

Arjen van den Berg

Sam Stepnowski

Jon Zmuda

Klaus Gawrisch

Clemens Glaubitz

Affiliations

Soon will be listed here.

Abstract

Tremendous progress has been made in determining the structures of G-protein coupled receptors (GPCR) and their complexes in recent years. However, understanding activation and signaling in GPCRs is still challenging due to the role of protein dynamics in these processes. Here, we show how dynamic nuclear polarization (DNP)-enhanced magic angle spinning nuclear magnetic resonance in combination with a unique pair labeling approach can be used to study the conformational ensemble at specific sites of the cannabinoid receptor 2. To improve the signal-to-noise, we carefully optimized the DNP sample conditions and utilized the recently introduced AsymPol-POK as a polarizing agent. We could show qualitatively that the conformational space available to the protein backbone is different in different parts of the receptor and that a site in TM7 is sensitive to the nature of the ligand, whereas a site in ICL3 always showed large conformational freedom.

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References

Yeliseev A . Expression and Preparation of a G-Protein-Coupled Cannabinoid Receptor CB for NMR Structural Studies. Curr Protoc Protein Sci. 2019; 96(1):e83. PMC: 6579622. DOI: 10.1002/cpps.83. View

Berger C, Ho J, Kimura T, Hess S, Gawrisch K, Yeliseev A . Preparation of stable isotope-labeled peripheral cannabinoid receptor CB2 by bacterial fermentation. Protein Expr Purif. 2010; 70(2):236-47. PMC: 2827653. DOI: 10.1016/j.pep.2009.12.011. View

Clark L, Dikiy I, Rosenbaum D, Gardner K . On the use of Pichia pastoris for isotopic labeling of human GPCRs for NMR studies. J Biomol NMR. 2018; 71(4):203-211. PMC: 7282444. DOI: 10.1007/s10858-018-0204-3. View

Sauvee C, Rosay M, Casano G, Aussenac F, Weber R, Ouari O . Highly efficient, water-soluble polarizing agents for dynamic nuclear polarization at high frequency. Angew Chem Int Ed Engl. 2013; 52(41):10858-61. DOI: 10.1002/anie.201304657. View

Munro S, Thomas K . Molecular characterization of a peripheral receptor for cannabinoids. Nature. 1993; 365(6441):61-5. DOI: 10.1038/365061a0. View

Jo S, Lim J, Klauda J, Im W . CHARMM-GUI Membrane Builder for mixed bilayers and its application to yeast membranes. Biophys J. 2009; 97(1):50-8. PMC: 2711372. DOI: 10.1016/j.bpj.2009.04.013. View

Gomez-Blanco J, Kaur S, Strauss M, Vargas J . Hierarchical autoclassification of cryo-EM samples and macromolecular energy landscape determination. Comput Methods Programs Biomed. 2022; 216:106673. DOI: 10.1016/j.cmpb.2022.106673. View

Wu J, Ma Y, Congdon C, Brett B, Chen S, Xu Y . Massively parallel unsupervised single-particle cryo-EM data clustering via statistical manifold learning. PLoS One. 2017; 12(8):e0182130. PMC: 5546606. DOI: 10.1371/journal.pone.0182130. View

Howlett A, Abood M . CB and CB Receptor Pharmacology. Adv Pharmacol. 2017; 80:169-206. PMC: 5812699. DOI: 10.1016/bs.apha.2017.03.007. View

10.

Vukoti K, Kimura T, Macke L, Gawrisch K, Yeliseev A . Stabilization of functional recombinant cannabinoid receptor CB(2) in detergent micelles and lipid bilayers. PLoS One. 2012; 7(10):e46290. PMC: 3463599. DOI: 10.1371/journal.pone.0046290. View

11.

Wang X, Liu D, Shen L, Li F, Li Y, Yang L . A Genetically Encoded F-19 NMR Probe Reveals the Allosteric Modulation Mechanism of Cannabinoid Receptor 1. J Am Chem Soc. 2021; 143(40):16320-16325. DOI: 10.1021/jacs.1c06847. View

12.

Pope A, Sanchez-Reyes O, South K, Zaitseva E, Ziliox M, Vogel R . A Conserved Proline Hinge Mediates Helix Dynamics and Activation of Rhodopsin. Structure. 2020; 28(9):1004-1013.e4. PMC: 7484155. DOI: 10.1016/j.str.2020.05.004. View

13.

Kriebel C, Asido M, Kaur J, Orth J, Braun P, Becker-Baldus J . Structural and functional consequences of the H180A mutation of the light-driven sodium pump KR2. Biophys J. 2022; 122(6):1003-1017. PMC: 10111219. DOI: 10.1016/j.bpj.2022.12.023. View

14.

Thurber K, Yau W, Tycko R . Low-temperature dynamic nuclear polarization at 9.4 T with a 30 mW microwave source. J Magn Reson. 2010; 204(2):303-13. PMC: 2874615. DOI: 10.1016/j.jmr.2010.03.016. View

15.

Xing C, Zhuang Y, Xu T, Feng Z, Zhou X, Chen M . Cryo-EM Structure of the Human Cannabinoid Receptor CB2-G Signaling Complex. Cell. 2020; 180(4):645-654.e13. PMC: 8247115. DOI: 10.1016/j.cell.2020.01.007. View

16.

Xiao Y, Ghosh R, Frederick K . In-Cell NMR of Intact Mammalian Cells Preserved with the Cryoprotectants DMSO and Glycerol Have Similar DNP Performance. Front Mol Biosci. 2022; 8:789478. PMC: 8824258. DOI: 10.3389/fmolb.2021.789478. View

17.

Vogel A, Bosse M, Gauglitz M, Wistuba S, Schmidt P, Kaiser A . The Dynamics of the Neuropeptide Y Receptor Type 1 Investigated by Solid-State NMR and Molecular Dynamics Simulation. Molecules. 2020; 25(23). PMC: 7727705. DOI: 10.3390/molecules25235489. View

18.

Kimura T, Yeliseev A, Vukoti K, Rhodes S, Cheng K, Rice K . Recombinant cannabinoid type 2 receptor in liposome model activates g protein in response to anionic lipid constituents. J Biol Chem. 2011; 287(6):4076-87. PMC: 3281699. DOI: 10.1074/jbc.M111.268425. View

19.

Lomize M, Lomize A, Pogozheva I, Mosberg H . OPM: orientations of proteins in membranes database. Bioinformatics. 2006; 22(5):623-5. DOI: 10.1093/bioinformatics/btk023. View

20.

Ge H, Wang H, Pan B, Feng D, Guo C, Yang L . G Protein-coupled Receptor (GPCR) Reconstitution and Labeling for Solution Nuclear Magnetic Resonance (NMR) Studies of the Structural Basis of Transmembrane Signaling. Molecules. 2022; 27(9). PMC: 9101874. DOI: 10.3390/molecules27092658. View