SCFFBXL3 ubiquitin ligase targets cryptochromes at their cofactor pocket

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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.

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Figure 1: Structure of murine CRY2 PHR in apo- and FAD-bound forms.
Figure 2: The open FAD-binding pocket of murine CRY2 PHR.
Figure 3: Overall structure of the CRY2–FBXL3–SKP1 complex.
Figure 4: Structure of the FBXL3 LRR domain.
Figure 5: Interaction between the FBXL3 C-terminal tail and the CRY2 FAD-binding pocket.
Figure 6: Structural and functional analyses of the FBXL3–murine CRY2 interface.

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Protein Data Bank

Data deposits

Structural coordinates and structural factors for FBXL3–CRY2–SKP1, CRY2–FAD and CRY2 are deposited in the Protein Data Bank under accession numbers 4I6J, 4I6G and 4I6E.


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We thank the beamline staff of the Advanced Light Source at the University of California at Berkeley for help with data collection, and members of the Zheng laboratory for discussion. This work is supported by the Howard Hughes Medical Institute (N. Z. and M. P.), the National Institutes of Health (R01-CA107134 to N.Z., 5T32-HL007151 to L.B., and R01-GM057587, R37-CA-076584 and R21-CA161108 to M.P.), and the University of Washington (S.T.M. and M.F.B.).

Author information

The protein purification and crystallization experiments were conceived by W.X., L.B., M.P. and N.Z., initiated by N.H.S., and conducted by W.X. W.X. and N.Z. determined and analysed the structures. FAD fluorescence and in vitro competition experiments were conceived by W.X., T.R.H. and N.Z., and conducted by W.X. and T.R.H. Mutational and binding studies, and stability analyses were conceived by L.B., M.P., W.X. and N.Z., and conducted by L.B. S.T.M. and M.F.B. conducted native mass spectrometry experiments.

Correspondence to Ning Zheng.

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The authors declare no competing financial interests.

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This file contains Supplementary Figures 1-12, Supplementary Methods, Supplementary Tables 1-2, a Supplementary Discussion and additional references. (PDF 7332 kb)

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Xing, W., Busino, L., Hinds, T. et al. SCFFBXL3 ubiquitin ligase targets cryptochromes at their cofactor pocket. Nature 496, 64–68 (2013) doi:10.1038/nature11964

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