UC Santa Cruz

Nearly all terrestrial organisms possess an intrinsic biological timekeeping system that functions to align key physiological processes with the solar day. In mammals, this circadian cycling is driven by temporally specific interactions between the heterodimeric transcription factor, CLOCK:BMAL1 and its cognate transcriptional regulators. The biochemical processes that drive the handoff between active and repressive complexes tunes the phase, period and robustness of circadian cycling. In this study, we show that the unstructured C-terminal transactivation domain (TAD) of BMAL1 is a regulatory hub where transcriptional activators and repressors compete for binding. Using NMR spectroscopy and real time monitoring of circadian cycling in mammalian cells we studied how interactions of the TAD with transcriptional regulators control the generation of circadian rhythms. We show that the KIX and TAZ1 domains of CBP and the CC helix of CRY1 interact with the BMAL1 TAD at overlapping sites. Perturbations of these regions of BMAL1 by site-directed mutagenesis have direct consequences for the balance of transcriptional activation and repression and elicit large changes the period of circadian oscillations. Using paramagnetic resonance spectroscopy we show that the BMAL1 TAD undergoes significant and differing conformational rearrangements upon binding CBP KIX and CRY CC. Furthermore, we identified a slow conformational change in the extreme C-terminus of BMAL1, a region of the TAD that is essential for normal circadian timekeeping. Using NMR spectroscopy, we performed structural and kinetic analysis of the conformational switch to identify a cis/trans isomerization about a Trp-Pro imide bond. Using NMR, fluorescence polarization and cell-based studies, we further characterized the switch to assess its role in circadian cycling and identified cyclophilins capable of regulating the rate of switch interconversion in vitro. Together, these data highlight the importance of structural dynamics of the BMAL1 TAD in tuning the molecular circadian clock.