The is a vital regulatory component of the circadian rhythm protein regulation system. CLOCK (Circadian Locomotor Output Cycles Kaput) and BMAL1 (Brain and muscle Arnt-like protein-1) are PER-ARNT-SIM (bHLH-PAS) proteins. (The structure highlighted in yellow is a portion of bHLH structure in CLOCK) The CLOCK:BMAL1 heterodimer is the main transcriptional activator in the mammalian circadian mechanism.[1] The binding between CLOCK and BMAL1 involves the N-terminal bHLH, PAS-A and PAS-B domains of both proteins. Each domain (bHLH, PAS-A, PAS-B) binds to its corresponding equivalent in the other protein. Though both proteins contain the same types of domains with similar primary amino acid sequences in each, the overall heterodimer is surprisingly asymmetrical due to differences in the spatial orientation of the domains in each protein. Since this heterodimer complex involves the binding of all of the major domains in both participating proteins, the overall binding affinity is very high.
Role in Circadian Rhythm
Circadian rhythms are operated by an endogenous core clock system that drives daily rhythms in behavior, physiology, and metabolism. In mammalian systems, the suprachiasmatic nucleus (SCN), which is located in the hypothalamus, is the locus of a master circadian clock. The SCN controls the expression of of proteins in a time dependent manner through a genetic feedback loop initiated by light passing through the eye.[2] The core molecular clockwork is composed of a transcriptional/post-translational feedback loop: CLOCK:BMAL1 (transcriptional activators) and PER:CRY (transcriptional repressors). In daytime, CLOCK and BMAL1 will form a heterodimer complex to bind the E-box promoter region of other circadian rhythm proteins, causing the transcription of Per (Period) and Cry (Cryptochrome). During the day, Per and Cry will dimerize and translocate into the nucleus, where they interact with CLOCK:BMAL1 to inhibit their own transcription, forming a negative feedback loop.[3] At night time, Per:Cry complex is degraded by a specific E3 ligase complex and the repression is relieved. After the repression level of Per:Cry is decreased, CLOCK:BMAL1 will be re-activated and start transcription again, forming the positive feedback loop. The entire negative/positive feedback loop take around 24 hours to complete, thus forming the core mechanism of the circadian clock in mammals.[4]
The Overall structure of CLOCK:BMAL1 complex
The is shown in the right. It is a tightly intertwined structure where CLOCK and BMAL1 are twisted together. Although the primary sequences of CLOCK and BMAL1 are similar, the structural arrangements of their domains are quite different.
CLOCK
is composed of three domains: one domain, two PAS domains ( and ). The connections between each domain are two . Compared to the flexible loops in BMAL1, the distances of the connection loops in CLOCK are longer.
BMAL1
is also composed of three domains: one domain, two PAS domains ( and ). There is a ~15-residue flexible loop () and a ~20-residue flexible loop () connecting between each domain.
The interface between CLOCK and BMAL1
In the formation of the CLOCK:BMAL1 heterodimer complex, each domain interacts with the corresponding domain of its partner subunit, i.e. CLOCK bHLH interacts with BMAL1 bHLH domain and so forth. In PSA domains, contain a five-stranded antiparallel β sheet and several α helices flanking the concave surface of the sheet. In those α helices, there are two packed in between the β-sheet faces of the two domains to mediate the heterodimeric PSA-A interactions.
PSA-A domain interface
The interface bin the CLOCK:BMAL1 PSA-A dimer is mainly facilitated by hydrophobic interactions. Specifically, Phe104, Leu105, and Leu113 on (Leu159, Thr285, Tyr287, Val315, and Ile317). Similarly, Phe141, Leu142, and Leu150 on BMAL1 PSA-A contact the hydrophobic β-sheet face of CLOCK (F122, I216, V252, and T254). As a result, many residues obtained in the CLOCK:BMAL1 interface are conserved among bHLH-PAS transcription factors. This result may indicate that CLOCK and BMAL1 have a common PSA-A domain dimerization mode.
PSA-B domain interface
The PSA-B domains of CLOCK and BMAL1 are stacked in parallel conformation. The β-sheet on PSA-B domain of BMAL1 contact the helical face of CLOCK PSA-B domain. The bury some hydrophobic residues on both subunits, including Try310, Val315, and Leu318 of CLOCK and Phe423, Trp427, and Val435 of BMAL1.
Fig. 1 The overlap structure of CLOCK:BMAL1 complex with bHLH Myc:Max-DNA complex (pdb: 1NKP). The figure is generated by Pymol
The binding interface between CLOCK:BMAL1 and E-box element
As shown in Fig. 1, the four-helical bHLH bundle in the CLOCK:BMAL1 heterodimer overlaps with the bHLH Myc:Max-DNA complex (pdb: 1NKP, in wheat). This four-helical bundle is highly hydrophobic, which indicating that dimerization of the bHLH domains should help stabilize the CLOCK:BMAL1 complex.[5] This bHLH conformation is also important for the E-box recognition.
The downstream effects of the altered circadian rhythm
It has been shown in recent years that people who have lifestyles which involve light exposure that is different than the normal 12 hours of daylight/12 hours of night (such as people who work the night shift, have insomnia, often work late) have significantly increased chances of developing cancer. The circadian rhythm is partially dictated by the amount of light the organism receives so behavioral changes will causes differential activation of circadian rhythm proteins. This indicates that disruption of the normal circadian rhythm gene regulation cycle has severe downstream effects on the host's genetic makeup. Additionally, it has been shown that cancer tissues often have distorted circadian rhythms, showing the significance of circadian rhythms to cancer progression. [6]
Mutated forms of CLOCK exist which do not regulate protein expression correctly and thereby result in altered circadian rhythms. CLOCK-delta19 is a mutant form of CLOCK which binds to BMAL1 normally but the resulting heterodimer does not activate transcription of certain other circadian rhythm proteins. Mutant mice carrying this altered CLOCK protein have shown abnormal circadian rhythms as a result. An example of the downstream effect of this mutant form of CLOCK is the resulting abnormal expression of NAMPT. In normal mice, NAMPT is expressed in a circadian manner, showing oscillations in expression regardless of light conditions. However, in mice with mutant CLOCK-delta19, NAMPT is not expressed in an circadian manner and the overall expression is lower. This leads to further issues as NAMPT is the rate limiting enzyme for the biosynthesis of NAD+, which is an important biological coenzyme.[7]
