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.
