Structural dynamics of a dimeric GPCR at the single molecule level – DynaMuR2

The four methods that will be used are:
1- the analysis of population FRET signals on receptors labeled on the surface of transfected cells by the trFRET approach
2- the development of several biosensors based on the use of unatural amino acids (UAA), and leading to distance variations at the time of conformational changes of the receptor
3- the extraction of receptors labelled with two fluorophores (donors and acceptors), in order to measure the FRET signal at the level of single, isolated molecules.
4- setting up a method to measure its FRET variation on single molecules in a cellular environment.

During the first 18 months we have:
1- validated several AANN-based biosensors, allowing to measure different conformational changes of the mGlu2 receptor by FRET
2- used the previously described biosensor to validate our approach in single molecules, and to find the conditions allowing to keep the full properties of the receptor.
3- Thanks to this approach, we showed that Glutamate was a partial agonist, and that the maximal activity of the receptor could only be obtained in the presence of a positive allosteric modulator, either a small molecule, or the G protein
4- Using this approach, we were able to demonstrate that a nanobody could activate the mGlu4 receptor as well as glutamate.
5- determined the structure of the mGlu5, GABAB, mGlu2, mGlu7 and mGlu2-7 receptor, in different conformational states.

Now that we have set up the biosensors, and validated the experimental approach, we will be able to study the different conformational changes in the different domains of the receptor, in order to understand how these changes are coordinated.
These studies will be carried out on the homodimeric receptors mglu2 and mGlu5, as well as on heterodimeric receptors which we have shown to exist in the brain

No patent has been filed in the framework of this program to date.
Seven publications have been published during the first 18 months of this project, in major international journals:

1. Nasrallah C, Cannone G, Briot J, Rottier K, Berizzi AE, Huang C-Y, Quast RB, Hoh F, Banères J-L, Malhaire F, Berto L, Dumazer A, Font-Ingles J, Gómez-Santacana X, Catena J, Kniazeff J, Goudet C, Llebaria A, Pin J-P, Ragunath VK, Lebon G (2021) Agonists and allosteric modulators promote signalling from different metabotropic glutamate receptor 5 conformations. Cell Reports: 2021;36:109648. doi: 10.1016/j.celrep.2021.109648.
2. Cao A-M, Quast RB, Fatemi F, Rondard P, Pin J-P, Margeat E (2021) Allosteric modulators enhance agonist efficacy by increasing the residence time of a GPCR in the active state. bioRxiv. DOI 10.1101/2021.01.05.424557
3. Cao A-M, Quast RB, Fatemi F, Rondard P, Pin J-P, Margeat E (2021) Allosteric modulators enhance agonist efficacy by increasing the residence time of a GPCR in the active state. Nat Commun 12:5426.DOI 10.1038/s41467-021-25620-5
4. Haubrich J, Font J, Quast RB, Goupil-Lamy A, Scholler P, Nevoltris D, Acher F, Chames P, Rondard P, Prézeau L, Pin J-P (2021) A nanobody activating metabotropic glutamate receptor 4 discriminates between homo and heterodimers. Proc Natl Acad Sci (USA) 118:e2105848118.
5. Shen C, Mao C, Xu C, Jin N, Zhang H, Shen D-D, Shen Q, Wang X, Hou T, Chen Z, Rondard P, Pin J-P, Zhang Y, Liu J (2021) Structural basis of GABAB receptor-Gi protein coupling. Nature 594:594-598.
6. Du J, Wang D, Fan H, Xu C, Tai L, Lin S, Han S, Tan Q, Wang X, Xu T, Zhang H, Chu X, Yi C, Liu P, Wang X, Zhou Y, Pin J-P, Rondard P, Liu H, Liu J, Sun F, Wu B, Zhao Q (2021) Structures of human mGlu2 and mGlu7 homo- and heterodimers. Nature 594:589-593.
7. Quast R.B., Margeat E. (2021) Single molecule FRET on its way to structural biology in live cells. Nature Methods – News and Views, doi.org/10.1038/s41592-021-01084-9.

This project aims at studying the structural dynamics of a dimeric G protein-coupled receptor (mGlu2) using innovative approaches allowing the study of single molecule at a time scale not yet achieved for this type of receptors.
GPCRs represent the main gene family in the human genome (3% of our genes). These receptors are expressed in every cell and are key elements in cell communication. Every cell expresses tens of these receptors allowing a fine-tuning of their activity. These receptors are the main target for therapeutic drugs and are still the main targets of R&D programs in pharmaceutical companies for the identification of novel therapeutic approaches in every clinical field.
Conceptual revolutions emerged recently regarding the properties of these receptors, such as the ability of some ligands to engage only a sub-set of the pathways activated by the targeted receptor. The most recent data indicate that this is likely the consequence of a differential control of the equilibrium dynamics between various conformational states, rather than the stabilization of a specific conformation. GPCRs are also reported to form dimers and larger oligomers composed of different GPCR subtypes. This latter property offers novel possibilities for allosteric interactions between receptors. It is therefore expected that the structural dynamics of one subunit affect that of its partner within GPCR dimers. However, GPCR dimerization remains a subject of intense debate in the field as such complexes in native cells and their functional roles remain elusive. This is due to the lack of our understanding of the basic mechanisms involved in the allosteric control between subunits and the lack of specific tools to control their activity in vivo.
Metabotropic glutamate receptors (mGluRs) represent the best model to study the structural dynamics of dimeric GPCRs, as these are constitutive and covalent dimers. Our goal is to analyze the structural dynamics of mGlu2R through a combination of innovative approaches, with the aim to better understand the structural bases of the allosteric interaction between subunits. We will measure the dynamics of the conformational changes of the various domains within each subunit at the single molecule level and at the microsecond time scale, as well as the relative movements between the subunits. These studies will be carried out under basal, fully inactive with bound inverse agonist, or active conditions. As such, we will bring important information for our understanding of how one subunit controls the conformational dynamics of the other.
Our project involves three groups located on the same campus in Montpellier: that of E Margeat (CBS), a biophysicist specialist of single molecule studies using smFRET; that of G Lebon (IGF), a structural biologist specialist in the thermostabilization of membrane proteins especially GPCRs: and that of JP Pin (IGF) a world leader in the GPCR field recognized for his expertise on mGlu and GABAB receptors.
Innovative approaches based on the thermostabilization of GPCRs, the analysis of the structural dynamics of GPCRs at the single molecule level using smFRET, and the use of in house developed new technologies to specifically label proteins using unnatural amino acids, will be used. These will pave the way for the experimental bases necessary for our understanding of the allosteric coupling between GPCRs and the control of their signaling properties.