Time-Resolved Native Mass Spectrometry Reveals Reversible Light-Driven Oligomerization of Arabidopsis Cryptochrome 1 and Its Antagonism by BIC1.

Cryptochromes (CRYs) are blue-light photoreceptors that mediate light-dependent signaling in plants. Here, we uncover the molecular mechanism underlying blue-light activation of the Arabidopsis thaliana cryptochrome 1 photolyase homology region (CRY1-PHR) using time-resolved native mass spectrometry combined with kinetic modeling. This approach enables direct monitoring of light-driven complex formation with temporal and molecular resolution. We show that blue-light activation of CRY1-PHR follows a reversible assembly pathway in which monomers rapidly form dimers that further assemble into tetramers. A quantitative two-step kinetic model captures the dynamic interplay between light-induced oligomerization and thermal disassembly. Strikingly, ATP accelerates tetramer formation and stabilizes oligomers by tuning the underlying photochemistry of the flavin adenine dinucleotide (FAD) chromophore. In contrast, the Blue-light Inhibitor of Cryptochromes 1 (BIC1) acts as a potent antagonist. BIC1 binds to CRY1-PHR even in the dark, with significantly increased affinity under blue light, thereby inhibiting oligomerization and actively disassembling pre-formed tetramers. This disassembly is light-independent and occurs regardless of CRY's redox state. Together, these findings provide a kinetic and mechanistic framework for reversible blue-light signaling by plant CRYs and highlight how opposing regulators precisely modulate photoreceptor activation at the molecular level.