SCFFBXL3 ubiquitin ligase targets cryptochromes at their cofactor pocket

The cryptochrome (CRY) flavoproteins act as blue-light receptors in plants and insects, but perform light-independent functions at the core of the mammalian circadian clock. To drive clock oscillations, mammalian CRYs associate with the Period proteins (PERs) and together inhibit the transcription of their own genes. The SCFFBXL3 ubiquitin ligase complex controls this negative feedback loop by promoting CRY ubiquitination and degradation. However, the molecular mechanisms of their interactions and the functional role of flavin adenine dinucleotide (FAD) binding in CRYs remain poorly understood. Here we report crystal structures of mammalian CRY2 in its apo, FAD-bound and FBXL3–SKP1-complexed forms. Distinct from other cryptochromes of known structures, mammalian CRY2 binds FAD dynamically with an open cofactor pocket. Notably, the F-box protein FBXL3 captures CRY2 by simultaneously occupying its FAD-binding pocket with a conserved carboxy-terminal tail and burying its PER-binding interface. This novel F-box-protein–substrate bipartite interaction is susceptible to disruption by both FAD and PERs, suggesting a new avenue for pharmacological targeting of the complex and a multifaceted regulatory mechanism of CRY ubiquitination. Crystal structures of mammalian CRY2, one of the cryptochrome flavoproteins that have light-independent functions at the core of the circadian clock, show that it binds FAD dynamically and that the F-box protein FBXL3 captures CRY2 by occupying its FAD-binding pocket and burying its PER-binding interface. Cryptochromes are photoreceptors found in both plants and animals and are required for the function of the circadian clock. In this study, Ning Zheng and colleagues present the first crystal structure of a mammalian cryptochrome protein, CRY2, in free and bound states. The structural analysis reveals novel and unexpected features when compared to other light-sensitive cryptochromes and establishes a framework for understanding how cryptochromes function in the clock pathway. Of particular interest is the mechanism by which the mammalian F-box protein FBXL3 captures CRY2, by occupying its FAD-binding pocket with a tryptophan-bearing C-terminal tail.