M. musculus Circadian
Rhythm Protein CLOCK:BMAL1
Spencer Byers '16 and Kyle Hardacker '15
Contents:
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.
CLOCK:BMAL1
DNA Binding
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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