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 elucidate the mechanism by which Arabidopsis CRY1 forms signalling-active tetramers in response to blue light using time-resolved native mass spectrometry combined with kinetic modelling. We found that oligomerization proceeds via a defined, reversible 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 amplifies this response, accelerating tetramer formation, stabilising oligomers and tuning the underlying photochemistry of the FAD chromophore. In contrast, the Blue-light Inhibitor of Cryptochromes 1 (BIC1) binds to CRY1 with nanomolar affinity under blue light conditions, acting as a potent antagonist by not only blocking oligomerization, but also actively dismantling pre-assembled tetramers. This process is light-independent and occurs regardless of CRY1’s 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.