Apo-state structure of the metabotropic glutamate receptor 5 transmembrane domain obtained using a photoswitchable ligand

Abstract

Metabotropic glutamate receptor 5 (mGlu5) is implicated in various neurodegenerative disorders, making it an attractive drug target. Although several ligand-bound crystal structures of mGlu5 exist, their apo-state crystal structure remains unknown. Here, we study mGlu5 structural changes using the photochemical affinity switch, alloswitch-1, in combination with time-resolved freeze-trapping methods. By X-ray crystallography, we demonstrated that isomerizing alloswitch-1 leads to its release from the binding pocket within a few seconds. The apo structure, determined at a resolution of 2.9 Å, is more comparable to the inactive state than to the active state. Our approach presents an accessible alternative to time-resolved serial crystallography for capturing thermodynamically stable transient intermediates. The mGlu5 apo-structure provides molecular insights into the ligand-free allosteric pocket, which can guide the design of new allosteric modulators.

Keywords: GPCR; X‐ray crystallography; apo‐state; light activation.

FIGURE 1

Photoisomerization of alloswitch‐1. (a) The chemical structures of alloswitch‐1. The azobenzene bond of alloswitch‐1 is highlighted in purple. (b) A structural superposition of the cis‐ and trans‐forms of alloswitch‐1 in the mGlu5 binding pocket. In the dark state, mGlu5 (gray) binds the trans‐form of alloswitch‐1 (cyan). The cis‐form of alloswitch‐1 (black) was generated by rotating the azo‐bond using Coot (Emsley et al., 2010). This speculative view illustrates the steric clash of the cis‐form with mGlu5, as experimental X‐ray data for the conformation are not available. (c) The set up for controlled illumination (i) using spitrobot and (ii) using an electronic cryo‐stream blocker. (d) Q‐weighted isomorphous difference peaks measured using the spitrobot, (e–g) and the annealing experiment. Q‐weighted difference electron density maps F _o(light) − F _o(dark) are contoured at 3.0 sigma and carved around alloswitch‐1. (h) The correlation between the activation time and the volume of the negative density around alloswitch‐1 in each X‐ray dataset. The datasets provided the q‐weighted difference maps shown in d–g are highlighted. For the comparison, the datasets obtained using the spitrobot are plotted at time zero and separated by the dotted line.

FIGURE 2

The apo‐state structure of mGlu5 TMD. (a) Overall structure of apo‐state mGlu5. Regions showing conformational changes (b) Phe788^6.53, (c) Trp785^6.50, and (d) TM5 are highlighted on the model. (b–d) Extrapolated map of local structural movements showing the apo‐state (cyan) and the dark state (gray) models. The extrapolated map is contoured at 1.5 sigma and carved 2.0 Å around structural models. (e) Highlighting the TM5 conformations among alloswith‐1 bound, apo, and active states (PDB ID: 6N51 chain B) (Koehl et al., 2019) by aligning the structures using TM1, 2, 3, 4, and 7.