NMR characterization of disordered transactivation domain in the C- terminus of BMAL1 circadian protein

Abstract

The core mechanism of the mammalian circadian clock features a translational transcriptional feedback loop, where the CLOCK-BMAL1 heterodimer activates transcription of clock controlled genes including the transcriptional repressors cryptochrome (CRY) and period (PER). Post heterodimerization with CLOCK, BMAL1 undergoes acetylation at lysine residue that is conserved (K537) in its C-terminus, and this modification occurs rhythmically within cells and plays a role in establishing the circadian rhythms. Acetylation at K537 enhances recruitment of CRY to the CLOCK BMAL1 complex which represses its own transcription. Mutation of this conserved lysine abolish circadian rhythms completely. However, molecular mechanism by which acetylation modulates affinities for binding of CRY remain unclear. Here we investigate the dynamic and structural features of the BMAL1 C-terminal domain (BCTD) using solution state NMR spectroscopy and try to find out how K537 acetylation affects protein function. We have expressed and purified murine BMAl1 CTD (residues 495-626) and prepared optimized protocols for achieving high purity protein. We have also characterized its structural and dynamic features. The intrinsically disordered C-terminus of BMAL1 displays limited chemical shift dispersion as expected of IDPs, howevevr relaxation measurements identify a dynamic hotspot in the region 604 – 615 exhibiting higher R2 rate constants inticating microsecond timescale conformational dynamics. This region is predicted to sample transient alpha helical conformations and harbours charged multiple residues that may mediate transient electrostatic interactions with K537. Our findings try to establish a foundation to understand acetylation dependent modulation of BMAL1 function in circadian timekeeping.