Photoswitch dissociation from a G protein-coupled receptor resolved by time-resolved serial crystallography

Abstract

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors in humans. The binding and dissociation of ligands tunes the inherent conformational flexibility of these important drug targets towards distinct functional states. Here we show how to trigger and resolve protein-ligand interaction dynamics within the human adenosine A_2A receptor. For this, we designed seven photochemical affinity switches derived from the anti-Parkinson's drug istradefylline. In a rational approach based on UV/Vis spectroscopy, time-resolved absorption spectroscopy, differential scanning fluorimetry and cryo-crystallography, we identified compounds suitable for time-resolved serial crystallography. Our analysis of millisecond-scale dynamics revealed how trans-to-cis isomerization shifts selected istradefylline derivatives within the binding pocket. Depending on the chemical nature of the ligand, interactions between extracellular loops 2 and 3, acting as a lid on the binding pocket, are disrupted and rearrangement of the orthosteric binding pocket is invoked upon ligand dissociation. This innovative approach provides insights into GPCR dynamics at the atomic level, offering potential for developing novel pharmaceuticals.

Fig. 1. Design and selection of synthetic photoswitches.

Derivatives were made through modifying the three basic building blocks of istradefylline: the xanthine group, the stilbene bridge, and the benzoyl side chain. Positions at which modifications were made are highlighted in yellow. These derivatives were subject to experimental characterization with three main properties deemed as important for time-resolved serial crystallography.

Fig. 2. Binding poses of istradefylline and five of its derivatives in the A_2A binding pocket.

The F_o-F_o omit maps are carved around the ligands and contoured at 3.0 sigma. Density of the omit maps indicates full replacement of theophylline by the photoswitches in the binding pocket.

Fig. 3. Biophysical characterization of photoswitch candidates.

A Differential scanning fluorimetry of photoswitches. The first derivative of the fluorescence curves (left) displays a progression of photoswitch-bound curves towards the apo curve after illumination. Increasing the illumination time (right) results in a progressive decrease in the melting temperature for all photoswitches: istradefylline, blue circles; StilSwitch1, black rightward triangles; StilSwitch2, red squares; StilSwitch3, yellow diamonds; StilSwitch4, green upwards triangles. Data are displayed as mean values ± the confidence interval calculated at 0.05 significance. Technical replicates were collected for each data point throughout multiple experiments using aliquots of the same protein sample. The number of replicates for each data point and the calculated confidence intervals are detailed in the source data file. The change in thermal shifts (ΔΔTm) of the photoswitches are: Istradefylline −4.04 °C, Switch2 −5.15 °C, Switch3 −8.67 °C, Switch4 −6.98 °C. B UV/Vis Absorption Spectra of photoswitches absorption under dark (solid line) and illuminated (dashed line) conditions in buffer (blue) and bond to the protein (yellow). All stilbene-derivatives display a ~6 nm shift in the trans absorption peak after protein binding. Source data are provided as a source data file.

Fig. 4. Photoswitch kinetics probed by transient absorption spectroscopy.

A–D: Transient absorption data of Switch3 in buffer (A) and with protein (C) on the left and the corresponding Lifetime Density Maps (LDM) on the right (B, D). For the transient data, positive signals indicate a product absorption (PA) or an excited state absorption (ESA), the negative signals refer to ground state bleach (GSB) or stimulated emission (SE). In the LDMs the positive components describe a rising negative or a decaying positive signal. The negative component depicts a decaying negative or a rising positive signal. In (E, F) normalized transients of Switch14 are displayed. The transients are normalized to the highest ESA signal in the dataset and show the maximum SE and remaining GSB signal, respectively. G shows the populations obtained from global target analysis for Switch3 bound to protein. Source data are provided as a source data file.

Fig. 5. Structural changes in the ligand binding pocket.

Changes observed in the A_2AR after isomerization of StilSwitch2 (A) and StilSwitch3 (B). Dark state (gray) and light state (purple) models are displayed overlayed with isomorphous difference maps (F_o(light)-F_o(dark), red, negative and green, positive at 3.0 sigma) carved around the light-activated switch. C View of the allosteric sodium binding site showing movement of the sodium ion after illumination and isomerization of Switch3 (purple) relative to the dark state (gray). D Even in the presence of the trans conformation of our istradefylline derivatives, the A_2AR ligand binding pocket is relatively open on the extracellular side. E In the case of Switch2, light activation widens the pocket but leaves the interaction between E169^ECL2 and H264^ECL3 intact. F In contrast the larger clash introduced by the extra methyl group has led to a shift of Switch3 towards the extracellular side and has widened the pocket by opening the interaction between E169^ECL2 and H264^ECL3.

Fig. 6. Time-resolved serial synchrotron crystallography.

A Pearson correlation using overlapping data bins shows a clear shift in states between 40–50 ms after photoactivation. Q-weighted isomorphous difference electron density maps (F_o(light)-F_o(dark), red, negative and green, positive contoured at 3.0 sigma) show the distribution of residue displacement across the whole protein. B, E Plot showing the integrated absolute density volume at 3.0 sigma in a 2 Å radius around each residue in the A_2AR. Gray boxes show the position of loops along the A_2AR protein and residues within 5 Å of the dark-state ligand are highlighted in blue. The density volume around the photoswitch is displayed in purple. C, F Model for changes observed in the binding pocket for state 1 (C) and state 2 (F). Dark state (gray) and light state (purple) models are displayed overlayed with q-weighted isomorphous difference maps (F_o(light)-F_o(dark), red, negative and green, positive at 3.0 sigma) carved around the highlighted residues and ligand. D, G View of the top of the A_2AR binding pocket for the two states. Source data are provided as a source data file.