Mechanistic insight into the oligomerization of Arabidopsis CRY1 and its inhibition by BIC1
Cryptochromes (CRY) convert blue light signals into biological responses, however, the molecular processes underlying their activation are not fully understood. In this study, we uncover the molecular mechanism underlying the blue-light activation of Arabidopsis CRY1 using time-resolved native mass spectrometry combined with kinetic modelling - an approach that allows us to monitor light-driven complex formation with temporal and molecular resolution. We show that CRY1 activation follows a defined, reversible assembly pathway in which monomers rapidly form dimers that then assemble into tetramers. A quantitative two-step model captures the dynamic interplay between light-driven assembly and thermal disassembly. Strikingly, ATP accelerates tetramer formation and stabilises oligomers by tuning the underlying photochemistry of the FAD chromophore. In contrast, the Blue-light Inhibitor of Cryptochromes 1 (BIC1) acts as a potent antagonist. We found that BIC1 binds to CRY1 even in the dark, with a significant increase in binding strength under blue-light conditions. BIC1 not only blocks CRY1 oligomerisation, but also actively dismantles pre-assembled tetramers. This disassembly process is light-independent and occurs regardless of CRY1s redox state. Together, these findings reveal a finely balanced regulatory system in which ATP and BIC1 act as opposing regulators to control CRY1 activation. This work provides a kinetic and mechanistic framework for reversible cryptochrome signalling and highlights how blue light responses can be precisely modulated at the molecular level.