- Home
- News
- Spotlight on Science
- Cryo-EM reveals...
Cryo-EM reveals the mechanism of positive allosteric modulation in mGlu5 receptor
21-01-2025
Cryogenic electron microscopy (cryo-EM) at beamline CM01 was used to determine the 3D structures of the metabotropic glutamate receptor mGlu5 bound to various positive allosteric modulators (PAMs). This study uncovers the diversity in PAM binding modes and their effects on receptor conformation and signalling output, providing a structural basis for the design of targeted therapies for neurological diseases such as schizophrenia.
Share
Communication between cells and their environment typically involves small molecules, with receptors acting as proteins that mediate signal transmission into cells. In the mammalian nervous system, L-glutamate is the primary excitatory neurotransmitter, playing a key role in synaptic plasticity as well as physiological processes like learning and memory. It binds to two types of receptors: ionotropic glutamate receptors, which act as ligand-gated ion channels (e.g., the N-methyl-D-aspartate (NMDA) receptor), and metabotropic glutamate receptors (mGlu), which are part of the G protein-coupled receptor (GPCR) family [1]. Aberrant glutamatergic transmission is associated with numerous neurological disorders, rendering these receptors significant therapeutic targets.
The mGlu receptor family consists of eight subtypes, divided into three groups based on pharmacology, sequence similarity, and cellular signalling pathways. In addition to glutamate, which is the orthosteric agonist, mGlu receptors can be modulated allosterically by molecules categorized as negative or positive allosteric modulators (NAM / PAM). Among these, mGlu5 plays an essential role in synaptic plasticity, and its dysfunction has been linked to neurological disorders such as Parkinson’s and Alzheimer’s diseases, making mGlu5 a validated drug target. Furthermore, positive allosteric modulation of mGlu5 holds therapeutic potential for mitigating symptoms of schizophrenia [2]. Despite its promise, designing specific PAMs for individual mGlu isoforms remains a challenging task.
The architecture of mGlu receptors is modular, featuring a large extracellular region composed of the Venus flytrap (VFT) domain, which binds the agonist L-glutamate, and a cysteine-rich domain (CRD) that connects the VFT to the transmembrane domain (7TM). Dimerization is crucial for mGlu receptor function, with receptors forming homo- and heterodimers. When L-glutamate binds to the VFT, it induces VFT closure, triggering a substantial conformational change conveyed to the 7TM domain via the CRD. This transition stabilizes an active state (see Movie), facilitating the optimal recruitment and binding of intracellular signalling partners such as G proteins [3-5].
Movie: Agonist binding to the VFT of class C GPCRs induces VFT closures and triggers conformational change conveyed to the 7TM domain via the CRD.
PAMs bind to the 7TM domain, approximately 100 Å from the L-glutamate binding site in the VFT, enhancing receptor signalling. The presence of multiple ligand-binding sites within the receptor can result in distinct signalling outcomes, a phenomenon known as biased signalling, which elicits diverse cellular responses. Understanding how PAMs bind and influence the dynamics of mGlu receptor activation is essential for designing novel therapeutic molecules.
This study utilized cryo-EM to determine the structures of detergent-purified, full-length mGlu5 bound to the high-affinity orthosteric agonist quisqualate (an L-glutamate analogue) and three distinct PAMs: VU0424465, VU0409551, and VU29. These structures revealed differential PAM binding modes and their effects on receptor conformation and activity.
The structure of mGlu5 bound to VU0424465, obtained using data from beamline CM01, provided direct access to the allosteric binding pocket, highlighting key residues involved in modulating receptor activity (Figures 1a and b). The PAMs VU0424465, VU0409551, and VU29 occupy a similar binding pocket in the 7TM domain but exhibit slight variations in their interactions, as confirmed through pharmacological characterization. These residues, previously implicated in stabilizing the inactive conformation of the 7TM domain, appear to interact differently with each PAM. Notably, PAMs and NAMs share a common binding site in mGlu5, with subtle differences influencing receptor activation.
Click figure to enlarge
Fig. 1: Single-particle cryo-EM analysis of the agonist- and PAM-bound mGlu5 receptor in detergent micelles. a) Averaged mGlu5 receptor particles obtained after 2D classification of PAM-bound mGlu5 receptors, computed from high-resolution cryo-EM images. b) 3D model of the mGlu5 receptor bound to the PAM VU0424465, showing one PAM molecule bound to each 7TM domain. The inset provides a zoomed-in view of the VU0424465 binding pocket, highlighting key interaction residues. c) Inactive state (Roo), where the antagonist LY1341494 is bound to the open VFT domain (PDB 7FD9). d) Intermediate activation state (Rcc) identified in this study, where quisqualate is bound to closed VFT domains but no PAM is present. The 7TM domains remain far apart, similar to the inactive Roo state. e) Active state (Acc), showing the receptor bound to quisqualate in the closed VFTs and VU0424465 in the 7TM domains. In the graphical representation of the mGlu5 receptor dimer, protomers are coloured green and blue, while ligands are depicted in sphere representation: LY341495 (orange), quisqualate (green and blue), and VU0424465 (yellow).
This study highlights the diversity of PAM binding modes and their effects on stabilizing the 7TM interface, a critical step for receptor activation and signalling. Analysis of a second dataset collected at beamline CM01 revealed an intermediate receptor state (Rcc; Figure 1d) between the inactive (Roo; Figure 1c) and active (Acc; Figure 1e) states. In the Rcc state, the VFTs, with agonist quisqualate bound, oscillate between discrete intermediate conformations. Additionally, there is interaction between the intracellular loops (ICL2) of each receptor’s 7TM domain, likely stabilizing the receptor’s conformation as a checkpoint before achieving the fully active state required for intracellular partner recruitment and signalling.
The structural characterization presented in this study underscores the impact of PAMs on receptor conformation, highlighting how they modulate the dimeric receptor’s activity. These findings provide valuable insights into the diversity of PAM binding and its effects on receptor activity, offering a structural framework for structure-based drug discovery programmes targeting mGlu5 PAMs.
Principal publication and authors
Conformational diversity in class C GPCR positive allosteric modulation, G. Cannone (a), L. Berto (b), F. Malhaire (b), G. Ferguson (b), A. Fouillen (b), S. Balor(c), J. Font-Ingles (d), A. Llebaria (d), C. Goudet (b), A. Kotecha (e), K.R. Vinothkumar (f), G. Lebon (b), Nat. Commun. 16, 619 (2025); https://doi.org/10.1038/s41467-024-55439-9. PMID: 39805839; PMCID: PMC11730304.
(a) MRC Laboratory of Molecular Biology, Cambridge (UK)
(b) IGF, Université de Montpellier, CNRS, INSERM, Montpellier (France)
(c) METi, Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS, Toulouse (France)
(d) MCS, Laboratory of Medicinal Chemistry & Synthesis, Department of Biological Chemistry, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona (Spain)
(e) Material and Structure Analysis Division, Thermo Fisher Scientific, Eindhoven (The Netherlands)
(f) National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK Post, Bengaluru (India)
References
[1] F. Sladeczek et al., Nature 317, 717-719 (1985).
[2] S. Dogra & P.J. Conn, Mol. Pharmacol. 101, 275-285 (2022).
[3] A. Koehl et al., Nature 566, 79-84 (2019).
[4] C. Nasrallah et al., Cell Rep. 36, 109648 (2021).
[5] K. Krishna Kumar et al., Nature 629, 951-956 (2024).
About the beamline: CM01 |
Beamline CM01 hosts the ESRF’s Titan Krios cryo-electron microscope (cryo-EM) for single particle experiments. It is equipped with a K3 direct electron detector, a Quantum LS imaging filter and a Volta phase plate. At the ESRF, cryo-EM complements macromolecular crystallography and BioSAXS as powerful techniques for protein structure determination. Using single-particle imaging, cryo-EM provides near-atomic resolution, achieving subnanometre-resolution structures of up to ~2 Å. It is particularly suited for studying larger proteins, complexes, and viruses, typically exceeding 150 kDa, that are inherently challenging to crystallize. Its advanced image analysis capabilities enable in silico classification of different conformation states of a sample, even when these states co-exist in solution. |