M. musculus Circadian Rhythm Protein CLOCK:BMAL1

Spencer Byers '16 and Kyle Hardacker '15


I. Introduction

The Mus musculus circadian rhythm protein CLOCK:BMAL1 is a heterodimer DNA binding protein involved in the transcription of several genes implicated in the circadian clock mechanisms in mammals. The CLOCK:BMAL1 proteins dimerize and through interactions with E-box regulatory sites activate many circadian rhythm genes, including Period (PER) and Cryptochrome (CRY) during light phases. The PER and CRY proteins accumulate and dimerize forming a PER:CRY repressor complex. The PER:CRY complex translocates to the nucleus during dark phases where they inhibit the CLOCK:BMAL1 heterodimer. This ebb and flow of CLOCK:BMAL1 and PER:CRY complexes give rise to the full circadian cycle through autoregulatory negative transcriptional feedback loops.

The CLOCK:BMAL1 heterodimer is also involved in transcriptional regulation of other genes that are associated with E-box sites.  Although many of these genes have not been fully characterized, studies have shown knockout of CLOCK:BMAL1 leads to loss of circadian rhythm as a whole as well as insulin resistance. Further research has shown that reintroduction of ectopic CLOCK:BMAL1 induces circadian rhythm.

One model for gene activation by CLOCK:BMAL1 binding proposes histone remodeling as a mechanism. Another mechanism suggests that CLOCK:BMAL1 recruits transcriptional factors to promote gene activation. Recent research has provided evidence for the former model of histone modification. 

II. General Structure

CLOCK:BMAL1 is a heterodimer formed by CLOCK and BMAL1. CLOCK is 361 amino acids long, and BMAL1 is 387 amino acids long. Each subunit contains one basic helix-loop-helix (bHLH) domain. The bHLH domains are involved in binding of the protein to DNA. Furthermore each subunit contains two PAS (period-ARNT-single-minded) domains, PASA and PASB . PASA and PASB of the BMAL1 subunit and PASA and PASB of the CLOCK subunit form the core of the CLOCK:BMAL1 heterodimer and are involved in dimerization. Although the domains' primary sequences are highly conserved the distribution of the domains varies distinctly leading to the asymmetric structure of the protein as a whole.

Each domain interacts with its respective counterpart on the other subunit leading to tightly a tightly entertwined heterodimer. Connecting the N-terminus of bHLH and C-terminus of the PASA domains in each heterodimer is a flexible loop (L1). In BMAL1, L1 is ~15 residues in length and is flexible making the alpha-helices continuous between the bHLH and PASA domains. L1 is very flexible with a high B-factor value. Therefore the L1 residues have not been fully characterized.

Conversely L1 in CLOCK is ~30 amino acids long and buried in the protein complex interface, making the loop much less solvent exposed and flexible. Due to the difference in loop structure, frequency, and distribution, CLOCK:BMAL1 forms an asymmetric protein dimer.

Electrostatic Potential of CLOCK:BMAL1 Heterodimer Faces

When the two subunits form the dimer, the heterodimer has two faces. The CLOCK and BMAL1 faces show different electrostatics. The CLOCK face, composed mostly of Beta-sheets in the PAS domains, is negative as opposed to the BMAL1 PASA face which is positive. The BMAL1 face is concave and the CLOCK face in convex allowing for the subunits to fit together.

III. Dimerization

The CLOCK:BMAL1 dimer assembles due to PASA interface between the two subunits. The dimer interface consists of hydrophobic interactions between the A'alpha helix of CLOCK and the Beta-sheets of BMAL1 and the A'alpha helix of BMAL1 and the Beta-sheets of CLOCK. Specifically when Ile317 is mutated to aspartic acid transcriptional activity is decreased by approximately 80% demonstrating this amino acid critical role in the hydrophobic interactions that give rise to dimerization at PASA.

Further dimerization interactions are found between the corresponding PASB domains in each subunit. Similar to PASA, hydrophobic interactions are present for dimer interface where non-polar amino acids are buried into the core of the protein, inaccessible to solvent molecules. Notably, Trp284 on the alpha helical face of CLOCK PASB and Trp427 on a loop transversing two Beta-sheets of PASB of BMAL1 form aromatic interactions due to ring stacking.

Finally, bHLH domains form a four-helical bundle that is highly hydrophobic at its core. These hydrophobic interactions indicate a dimerization site between CLOCK and BMAL1 at bHLH sites, further stabilizing the CLOCK:BMAL1 complex. Four leucine amino acids play an important role in the hydrophobic interactions that allow the dimerization in the bHLH sites. Research has shown Leu57 plays a critical role in the dimerization of the corresponding bHLH domains in CLOCK:BMAL1, creating the alpha helix forks necessary for DNA binding.

IV. DNA Binding

CLOCK:BMAL1 heterodimer binds DNA via the bHLH forks formed during dimerization. Specifically alpha helix 1 of the bHLH domains insert into the major grooves in the DNA duplex in order to recognize E-box sites through hydrogen bonding between serine residues and DNA. E-box sites are ~20 base pairs upstream of genes that they activate and their canonical motif is 5'-CACGTG-3'. Serine residues [Ser42 (CLOCK), Ser90 (BMAL1)] in alpha helix 1 are implicated in CLOCK:BMAL1 DNA binding through hydrogen bonding.

The serine residues are located near the periphery of alpha helix 1 making them highly accessible to DNA. When the serine residues were mutated to leucine residues DNA binding was decreased by ~50% emphasizing the critical role these amino acids play in CLOCK:BMAL1 DNA binding. Upon the binding of CLOCK:BMAL1 complex to an E-box site, transcription is upregulated. One proposed mechanism is through recruitment of histone acetyl transferases to decondense the nucleosome into heterochromatin allowing transcriptional machinery access to the DNA. An alternative mechanism that has been proposed is that the binding of CLOCK:BMAL1 recruits transcription factors to the E-box site, upregulating transcription of the target gene.


V. Cryptochrome:Period Binding

The CLOCK:BMAL1 complex activates transcription of both PER and CRY. Upon accumulation, PER and CRY proteins dimerize and translocate to the nucleus where they inhibit the activity of CLOCK:BMAL1. Trp362 of the CLOCK PASB domain is involved in the interaction with CRY:PER. Trp362 is located on a loop between Beta-sheets, similar to Trp427 in BMAL1 that interacts with CLOCK in the PASB domains that assists in dimerization. This location conveys a high degree of flexibility allowing the insertion of Trp362 into CRY:PER. Additionally G332, H360, Q361, and E367 assist CRY CLOCK:BMAL1 binding. These amino acids, along with Trp362 are exposed to the solvent and are readily available to interact with the CRY:PER protein complex, possibly through ring stacking.

VI. References

Bellet, MM and P. Sassone-Corsi. 2010. Mammalian circadian clock and metabolism - the epigenetic link. J Cell Sci 123: 3837-3848.

Bunge, MK, L.D. Wilsbacher, S.M. Moran, C. Clendenin, L.A. Radcliffe, et al. 2000. Mop3 is an essential of the master circadian pacemaker in mammals. Cell 103:1009-1017.

Huang, Nian, Yogarany Chellia, Yongli Shan Clinton A. Taylor, and Seung-Hee Yoo. 2014. Crystal Structure of the Heterodimeric CLOCK:BMAL1 Transcriptional Activator Complex. Science. 337:189-194.

Menet, J.S., S. Pescatore, and M. Rosbash 2014. CLOCK:BMAL1 is a pioneer-like transcription factor. Genes & Development 28:8-13.

Yoshitane, H, H. Ozaki, H. Terajima, N-H Du, Y. Suzuki, et al. 2014. CLOCK-Controlled Polyphonic Regulation of Circadian Rhythms through Canonical and Noncanonical E-Boxes. Molecular and Cellular Biology 34:1776-1787.

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