Structural basis of GPCR activation mechanism by using photoswitchable ligand, and X-ray diffraction. – SWITCH-ON

G Protein-Coupled Receptors (GPCRs) represent the largest family of membrane proteins. These key players in cell-to-cell communication are major drug targets, with more than 40% of marketed drugs targeting GPCRs. They are organised in 3 main classes, class A, B and C. The first structure of the full-length class C receptor dimer, the metabotropic glutamate receptor 5 (mGlu5), was recently reported. The structure highlights the molecular reorganisation of the extracellular domain and of the seven transmembrane domain (7TM) induced upon agonist binding. However, the molecular details of 7TM activation mechanism remain largely unknown. Here, we will investigate the structural dynamics of the mGlu5 receptor 7TM activation mechanism by using an azobenzene-containing photoswitchable ligands and the emerging method of serial crystallography.
Azobenzene-containing photoswitchable ligand are unique tools to explore the conformational dynamics of GPCRs activation mechanisms. Indeed, such light-controllable ligands can be rapidly, within ps timescale, and reversibly isomerised upon near UV light (trans-to-cis) and green light (cis-to-trans) exposure. In the present project, we will use alloswitch-1, an inverse agonist in its trans conformation that totally blocks the mGlu5 receptor activity through binding to the its 7TM. Upon exposure to UV-light, this compound switches to its inactive cis conformer, a neutral ligand that accumulates, but does not interfere with receptor activity and G protein activation. Thus, considering these two different alloswitch-1 conformations that can be controlled with light, we hypothesised that the in situ alloswitch-1 trans-to-cis isomerisation triggers conformational changes in the 7TM domain that unlock signal transduction and are involved in G protein activation.
We will combine mGlu5 photoswitchable inverse agonist, membrane protein biochemistry, macromolecular crystallography (MX), and static and time-resolved (TR) serial crystallography to decipher light-induced 7TM conformational changes and side-chain motions that define the structural basis of mGlu5 activity. The first objective of the project is to solve the structure of the mGlu5 7TM bound to the more stable conformation of the photoswitchable ligand, the trans isomer, by using MX at a micro focus beamline. The second objective is to obtain the structure of the mGlu5 7TM bound to the ligand in its cis-conformation by use of serial synchrotron crystallography (SSX). A third objective is to characterise the structural changes that underlie the allosteric modulation of the mGlu5 receptor by collection of pump-probe crystallographic data using serial femtosecond crystallography (SFX). Trans-to-cis isomerisation of alloswitch-1 will be triggered by exposure to a femtosecond UV-laser pulse (pump), and SFX data (probe) collected at various time pump-probe delays. As fourth and last objective, we will complete our structural investigation by performing a photopharmacological characterisation of the mGlu5 mutants in order to highlight the role in ligand binding and signal transduction of residues side chains identified in the high-resolution static and TR structures.
Data acquired within the framework of the SWITCH-ON project will inform on the conformational changes elicited by trans-to-cis isomerisation of alloswitch-1 in the mGlu5 7TM domain, offering a new structural basis for mGlu5 and other GPCR activation mechanism. The project will also provide the first demonstration that atomic-level insights into structural dynamics of proteins can be attained by combined use of diffusible azobenzene-containing photoswitchable ligand and serial crystallography. Finally, mGlu5 dysfunction is associated to neurologic disorders such as Fragile X syndrome and it has therefore been validated as a potential drug target. Our project will open doors to structure-based drug design taking into account the knowledge of mGlu5 7TM structural dynamics.