The circadian rhythm in mammals is regulated by various proteins whose activities are controlled by protein degradation and metabolism. Recent studies have shown that ubiquitin E3 ligases and metabolites such as flavin adenine dinucleotide (FAD) directly bind certain circadian clock proteins and regulate their functions. Here we use native mass spectrometry (MS) to probe the solution structures of a complex between an ubiquitin E3 ligase, SCFFBXL3-SKP1, and a mammalian cryptochrome, CRY2. Native MS of SCFFBXL3-SKP1-CRY2 reveals a 128 kDa heterotrimer with a bimodal charge-state distribution, suggesting the presence of two conformational families in solution. Collision cross section (CCS) values for that complex were determined using a Waters Synapt G2 HDMS. CCS values measured for the lower charge-state ions were indistinguishable from values calculated for a recently reported X-ray crystal structure (Xing et al. Nature DOI: 10.1038/nature11964). CCS values measured for the higher charge-state ions were 25-30 % larger, suggesting the presence of a discrete extended conformer. The presence of the extended conformer is suppressed upon incubation with FAD, even though mammalian CRY2 binds FAD very weakly (kD ~ 40 �M, ibid). This effect persists for several hours after FAD is removed via buffer exchange. The presence of an additional conformer of SCFFBXL3-SKP1-CRY2 and its depletion in the presence of FAD suggests that transient interactions with metabolites may regulate the complex via a long-lived conformational switch.

Ion mobility mass spectrometry of a mammalian circadian clock protein complex reveals a ligand-dependent conformational switch

246th National Meeting (2013)

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