Room-temperature light-state structure of the AsLOV2-Rac1 optogenetic actuator by sheet-on-sheet serial femtosecond crystallography

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.