Oral Presentation 50th Lorne Proteins Conference 2025

Dissecting GPCR function through nanodiscs; structural and pharmacological approaches (115078)

Wessel A C Burger 1 2 , Christopher J Draper-Joyce 3 , Susovan Das 1 , Hariprasad Venugopal 4 , Vi Pham 3 , Gunnar Schulte 5 , Arthur Christopoulos 2 3 , Celine Valant 3 , David M Thal 2 3 , Alisa Glukhova 1 2 6
  1. Structural Biology Division, Walter and Eliza Hall of Medical Research, Parkville, VIC, Australia
  2. ARC Centre for Cryo-electron Microscopy of Membrane Proteins, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Vic, Australia
  3. Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Australia
  4. Ramaciotti Centre for Cryo-electron Microscopy, Monash University, Clayton, Victoria, Australia
  5. Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
  6. Department of Biochemistry and Pharmacology, University of Melbourne, Melbourne, Victoria, Australia

G protein-coupled receptors (GPCRs) are integral membrane proteins crucial for cellular signalling and are prominent drug targets1. However, understanding GPCR structure and function in complex cellular environments can be challenging due to confounding factors such as dimerization, endogenous ligands and interactions with intracellular proteins. To address this, GPCRs must be studied in a controlled, reductionist environment where these factors are minimized. Traditional approaches, like detergent solubilization, can isolate GPCRs but represent a non-native environment and destabilize GPCRs2. Nanodiscs are a promising alternative as they provide a stable, membrane-like environment through a lipid bilayer encapsulated by a scaffold protein3. This setup preserves GPCR stability, structure, and function, allowing for a more accurate exploration of GPCR pharmacology and structure.  

Using MSP1D1 as a scaffold protein, we reconstituted two highly conserved GPCRs, the M2 and M4 muscarinic acetylcholine receptors (mAChRs) into nanodiscs. We examined high-affinity agonist binding at these GPCR nanodiscs in the presence of purified G proteins which represent the intracellular signalling partner of GPCRs. High-affinity binding can be readily observed in GPCR nanodiscs as the presence of GTP/GDP nucleotides and G proteins that both control the high-affinity state can be controlled for4. Our findings show that the clinical anti-psychotic agonist xanomeline is functionally selective for the M4 mAChR as it selectively stabilizes the high-affinity state at the M4 mAChR but not at the M2 mAChR5. By mutating non-conserved residues in the binding site and examining these mutants in nanodiscs, we reversed xanomeline’s selectivity, shifting its high-affinity binding from the M4 to the M2 mAChR. 

Allosteric modulators represent compounds that bind to a spatially distinct allosteric site6. Similar to G proteins, allosteric modulators can also promote high-affinity agonist binding, yet how this occurs has remained unknown. By examining allosteric modulators and G proteins at M2 mAChR nanodiscs, we show that allosteric modulators stabilise the lifetime of the high-affinity state that, in the presence of nucleotides, leads to an enhanced rate of initial signalling7. Overall, nanodiscs have provided fresh insights into GPCR function, revealing how clinical drugs selectively target even the most conserved GPCRs and showing how allosteric modulators work in concert with agonists and G proteins to enhance GPCR signalling

Nanodiscs can also enable structural characterisation of GPCRs in a native-like lipid environment. Our recent 2.6 Å cryo-electron microscopy (cryo-EM) structure of the GPCR Frizzled 7 in complex with a G protein within a detergent micelle revealed a well-resolved density at the interface of the receptor and G protein that has not been observed before. Due to this density, Frizzled 7 and the G protein display a unique conformation not seen at other GPCR-G protein complexes. To rule out detergent artifacts, we reconstituted the Frizzled 7-G protein complex into MSP1D1 nanodiscs and determined a 2.4 Å cryo-EM structure. This nanodisc-based structure confirmed the presence of a similar density at the receptor-G protein interface, which we propose to be a lipid molecule retained during purification, essential for complex stability. This discovery may point to a potential new role for lipids in stabilizing GPCR-G protein interfaces and regulating G protein coupling and signalling. Structurally, nanodiscs have validated the existence of a unique density at the GPCR-G protein interface, offering a new perspective and further understanding on GPCR – G protein interactions. Current and future work aims to identify this lipid and clarify its functional role in Frizzled 7 signalling.

  1. Hauser, A., Attwood, M., Rask-Andersen, M., et al. Trends in GPCR drug discovery: new agents, targets and indications. Nat Rev Drug Discov 16, 829–842 (2017).
  2. Tate, C.G. Practical Considerations of Membrane Protein Instability during Purification and Crystallisation. In: Mus-Veteau, I. (ed.) Heterologous Expression of Membrane Proteins. Methods in Molecular Biology™, vol 601 (2010).
  3. Bayburt, T.H., Grinkova, Y.V., Sligar, S.G. Self-Assembly of Discoidal Phospholipid Bilayer Nanoparticles with Membrane Scaffold Proteins. Nano Lett 2, 8, 853–856 (2002).
  4. Whorton, M.R., Bokoch, M.P., Rasmussen, S.G.F., Huang, B., Zare, R.N., Kobilka, B., Sunahara, R.K. A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein. PNAS 104 (18), 7682–7687 (2007).
  5. Powers, A.S., Pham, V., Burger, W.A.C., Thompson, G., Laloudakis, Y., Barnes, N.W., Sexton, P.M., Paul, S.M., Christopoulos, A., Thal, D.M., Felder, C.C., Valant, C., Dror, R.O. Structural basis of efficacy-driven ligand selectivity at GPCRs. Nat Chem Biol 19(7), 805–814 (2023).
  6. Burger, W.A.C., Sexton, P.M., Christopoulos, A., Thal, D.M. Toward an understanding of the structural basis of allostery in muscarinic acetylcholine receptors. J Gen Physiol 150(10), 1360–1372 (2018).
  7. Burger, W.A.C., et al. Positive allosteric modulation of a GPCR ternary complex. Sci Adv 10, eadp7040 (2024).