Room-temperature light-state structure of the AsLOV2-Rac1 optogenetic actuator by sheet-on-sheet serial femtosecond crystallography
- 1Jagiellonian University
Abstract
Light-oxygen-voltage (LOV) domains are blue-light sensors that make fused effector proteins optically controllable. The AsLOV2-Rac1 fusion (PA-Rac1) is one of the most widely used of these tools, yet the structural basis of its activation has been inferred largely from cryogenic structures and from solution studies, mostly of the isolated AsLOV2 domain. Here, room-temperature dark and light-state structures of AsLOV2-Rac1 at 1.8 [A] resolution are reported, determined by serial femtosecond crystallography at the SwissFEL Cristallina beamline with sheet-on-sheet (SOS) chip sample delivery. Repeated 450 nm pump-laser excitation during chip rastering accumulated the FMN-cysteine adduct in about 80 % of the molecules, giving strong Fourier difference maps. To our knowledge, this is the first light-state structure of PA-Rac1 or of any AsLOV2 fusion and the first AsLOV2 light state in which the thioether adduct is not compromised by X-ray radiolysis. Apart from the N-terminal residue and one marginal Rac1 loop residue, C displacements above 0.3 [A] between dark and light models recorded at the same temperature are confined to the chromophore environment, the D helix and the G{beta}-H{beta} and H{beta}-I{beta} loops of AsLOV2. These changes, including the Ile445 rotamer switch, are also seen in light-state structures of the isolated AsLOV2 domain, but the Asn414 rotation reported there is absent in the fusion. Comparison with the cryogenic dark-state structure also shows temperature-dependent differences that exaggerate the apparent changes. In the crystal, the GTP, the Mg2+ site and the switch regions of Rac1 show no detectable light-induced change and the J helix does not unfold, although transient or low-population states cannot be excluded. This modest response is consistent with the small difference in effector affinity between dark- and lit-state mutants in solution, with lattice contacts and an interdomain Ca2+ ion as possible further contributions.
Related
- Actuator Transcription Factors Resolve Combinatorial Binding Into Productive Gene Expression : An Evolutionary and Functional Framework for Gene Activation by Combinatorial TF Assemblies
- Symmetry Breaking under Vectorial Perturbations in Macromolecular X-ray Crystallography
- Correcting Multiple Scattering Enables Dynamical Electron Crystallography in Standard Workflows
- Antibody CDR-H3 loop flexibility: Insights from X-ray crystallography, structural bioinformatics, and the limits of current deep learning methods
- Sample-efficient time-resolved X-ray crystallography with serial small wedges
BibTeX
@misc{pachota2026roomtemperature,
title = {{Room-temperature light-state structure of the AsLOV2-Rac1 optogenetic actuator by sheet-on-sheet serial femtosecond crystallography}},
author = {M. Pachota and M. Wisniewska and M. K. Shankar and M. V. Appleby and A. Koczurowska and A. Asghar and M. Kepa and R. Ke and A. Solecka and T. Duran Kobylecka and B. Olasz and S. Mous and J. H. Beale and G. Gotthard and P. Nogly},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.27.762267},
url = {https://www.biorxiv.org/content/10.64898/2026.10.27.762267v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Room-temperature light-state structure of the AsLOV2-Rac1 optogenetic actuator by sheet-on-sheet serial femtosecond crystallography
AU - M. Pachota
AU - M. Wisniewska
AU - M. K. Shankar
AU - M. V. Appleby
AU - A. Koczurowska
AU - A. Asghar
AU - M. Kepa
AU - R. Ke
AU - A. Solecka
AU - T. Duran Kobylecka
AU - B. Olasz
AU - S. Mous
AU - J. H. Beale
AU - G. Gotthard
AU - P. Nogly
PY - 2026
DA - 2026/10/27
PB - bioRxiv
DO - 10.64898/2026.10.27.762267
UR - https://www.biorxiv.org/content/10.64898/2026.10.27.762267v1
AB - Light-oxygen-voltage (LOV) domains are blue-light sensors that make fused effector proteins optically controllable. The AsLOV2-Rac1 fusion (PA-Rac1) is one of the most widely used of these tools, yet the structural basis of its activation has been inferred largely from cryogenic structures and from solution studies, mostly of the isolated AsLOV2 domain. Here, room-temperature dark and light-state structures of AsLOV2-Rac1 at 1.8 [A] resolution are reported, determined by serial femtosecond crystallography at the SwissFEL Cristallina beamline with sheet-on-sheet (SOS) chip sample delivery. Repeated 450 nm pump-laser excitation during chip rastering accumulated the FMN-cysteine adduct in about 80 % of the molecules, giving strong Fourier difference maps. To our knowledge, this is the first light-state structure of PA-Rac1 or of any AsLOV2 fusion and the first AsLOV2 light state in which the thioether adduct is not compromised by X-ray radiolysis. Apart from the N-terminal residue and one marginal Rac1 loop residue, C displacements above 0.3 [A] between dark and light models recorded at the same temperature are confined to the chromophore environment, the D helix and the G{beta}-H{beta} and H{beta}-I{beta} loops of AsLOV2. These changes, including the Ile445 rotamer switch, are also seen in light-state structures of the isolated AsLOV2 domain, but the Asn414 rotation reported there is absent in the fusion. Comparison with the cryogenic dark-state structure also shows temperature-dependent differences that exaggerate the apparent changes. In the crystal, the GTP, the Mg2+ site and the switch regions of Rac1 show no detectable light-induced change and the J helix does not unfold, although transient or low-population states cannot be excluded. This modest response is consistent with the small difference in effector affinity between dark- and lit-state mutants in solution, with lattice contacts and an interdomain Ca2+ ion as possible further contributions.
ER -