Beale, J. H. | Search bioRxiv

bioRxiv2025

Time-resolved structures of β2-adrenergic receptor modulation by a photoswitchable beta-blocker

G protein-coupled receptors (GPCRs) regulate essential physiological responses and are important drug targets, yet their ligand-induced conformational dynamics remain poorly understood. The {beta}2-adrenergic receptor ({beta}2AR) is a prominent member of the GPCR family. It regulates bronchial and vascular function and is a significant drug target, particularly in respiratory and smooth muscle-related disorders. We employed time-resolved crystallography at X-ray free-electron lasers (XFELs) to capture the conformational dynamics of {beta}2AR bound to photoazolol-1, a beta-blocker derivative developed for photopharmacological applications. Structural snapshots of the receptor bound to trans-photoazolol-1 (pre-photoconversion), a strained intermediate, and the fully photoisomerized cis-photoazolol-1 reveal an intricate interplay between ligand chemistry and receptor plasticity. Isomerization of the azobenzene moiety induces distinct conformational changes within the orthosteric pocket, altering interactions with the extracellular loop 2 and transmembrane helices 5 and 6. Supported by functional assays, these structural shifts suggest that photoazolol-1 transitions from an inverse agonist to a neutral antagonist upon photoactivation. Our findings uncover a mechanism of GPCR modulation reminiscent of rhodopsin activation and offer a framework for designing ligands that harness light-driven transitions to achieve spatiotemporal control of receptor function.

biochemistry↗

bioRxiv2023

Fixed-target time-resolved crystallography at XFELs: the scourge of light contamination but reduced sample consumption

X-ray free electron laser (XFEL) light sources have allowed for the rapid growth of time-resolved structural experiments, which provide crucial information on the function of biological machines and their mechanisms. We set out to commission the SwissMX fixed-target sample delivery system at the SwissFEL Cristallina experimental station using the PSI developed MISP-chip for pump-probe time-resolved experiments. To characterise the system, we used the light-sensitive protein crystals of the Light-Oxygen-Voltage domain 1 (LOV1) from Chlamydomonas reinhardtii. Using different experimental settings, the adjacent-well light contamination was carefully assessed, indicating that it is crucial to control the light scattering from solid supports otherwise significant contamination can occur. However, our results show that, after the initial experiments and parameter refinement, the opaque MISP-chips are suitable for pump-probing a light-sensitive protein. This crystallographic experiment also probed the sub-millisecond structural dynamics of the LOV1 and indicated that at {Delta}t=10 s the covalent thioether bond is already established between the reactive Cys57 and FMN cofactor. This experiment validated the crystals to be suitable for in-depth follow up studies of the still poorly understood signal transduction mechanism. Importantly, the fixed-target delivery system also permitted a tenfold reduction in protein sample consumption compared to the most successful system used at XFEL, the high-viscosity extruder. This development creates the prospect of an exciting increase in XFEL project throughput for the field.

biophysics↗