CLOCK:BMAL1

Brad Clegg '20 and Hannah Sklar '20


Contents:


I. Introduction

Model View:


Many important physiological and developmental processes have basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) proteins as critical regulators of gene expression networks, but most of these structures are very poorly characterized. In order to form a functional DNA binding domain, these bHLH proteins must dimerize, with the bHLH-PAS proteins forming a specific dimer that is conferred by their PAS homology domains (Kewley et al., 2004).

The mammalian circadian clock runs by an autoregulatory transcriptional feedback mechanism. The bHLH-PAS proteins, CLOCK and BMAL1, play a key role in this mechanism as transcriptional activators (Huang et al., 2012). CLOCK and BMAL1 drive cadenced gene expression by rhythmically activating the expression of their respressors, PER and CRY, enhancing the transcription of Period and Cryptochrome genes during the daytime (Menet et al., 2014). At night, the proteins coded for by these genes, PER and CRY, build up, dimerize, and translocate into the nucleus to associate with the CLOCK:BMAL1 heterodimer, repressing further transcription. The PER:CRY repressor is eventually broken down by ligase complexes, allowing the CLOCK:BMAL1 complex to stimulate transcription again, starting the new circadian cycle. This reiterative process in the mammalian circadian clock repeats roughly every 24 hours. 

Core and N-Terminal Tail

Figure 1: Repressors PER and CRY interact with CLOCK:BMAL1 heterodimer at night and not during the day, creating the circadian rhythm in mammals (Sahar and Sassone-Corsi, 2009).


II. General Structure




In order to obtain a protein complex stable enough for crystallographic analysis, Huang et al. used protein constructs containing two tandem mouse BMAL1 (PAS-A and PAS-B) and CLOCK (PAS-A and PAS-B) and a domain for each subunit (BMAL1 bHLH and CLOCK bHLH). Looking at the three-dimensional structure of the CLOCK:BMAL1 complex, there is a tightly intertwined heterodimer with the three domains in CLOCK interacting with their partner domains in BMAL1. Although the three domains have very comparable amino acid sequences with their respective partner domain in the other subunit, there are drastic conformational differences between the different domains in their spatial relationship. This makes it so that this is an unusually asymmetric heterodimer.

This asymmetry is shown in multiple structural properties of the heterodimer, with one being that the BMAL1 bHLH alpha-2 helix is arranged with the A'alpha helix of the BMAL1 PAS-A domain. This is very different in the CLOCK subunit, which has a between the end of the alpha-2 helix of bHLH and the start of the A'alpha helix of PAS-A, and is connected by a flexible linker. This results in direct contact between PAS-A and the alpha-2 heix of bHLH in CLOCK, while there is no direct contact between the same domains in BMAL1.

The asymmetry is also shown by the electrostatic potential of the two subunits. BMAL1 has an overall positive electrostatic potential while that of the CLOCK subunit is overall negative. This is consistent with the exposed faces of the subunits, since the exposed regions of BMAL1 display a positive or neutral electrostatic potential while CLOCK has faces with a negative electrostatic potential. This source of asymmetry adds an important property governing the potential intereactions of the dimer and explains why the PER1, PER2, CRY1, and CRY2 proteins can bind differentially with the CLOCK and BMAL1 subunits.  

Core and N-Terminal Tail

Figure 2: Electrostatic potentials of the exposed faces of the CLOCK:BMAL1 heterodimer. Positive potentials are indicated by blue ovals and negative potentials are indicated by red ovals (Huang et al., 2012).


III. DNA Binding

In order for the heterodimer to act as a transcriptional activator, it must be able to bind DNA. In order to confirm this, the researchers used oligonucleotides containing th E-box sequence 5' CACGTG 3' from the mPer1 and mPer2 promoters and verified that binding to the protein complex occurred (Huang et al., 2014). Perhaps the most important part of the heterodimer in terms of DNA binding is the formed with the C-terminal halves of the alpha-1 helices collectively with the alpha-2 helices of both CLOCK and BMAL1 bHLH domains. Like in other bHLH proteins, dimerization of the complex helps to stabilize the hydrophobic core of this bundle. In order to recognize the E-box DNA, it is also essential for the bHLH domains to be in the correct conformation. To ensure precise interaction with the major groove sites of the E-box DNA duplex, the alpha-1 helices must be exactly aligned. The importance of this interaction is shown through a mutation of the hydrophobic core CLOCK L57 and L74, BMAL1 L95 and L115 to E, which completely abolished the transactivation activity of the full length CLOCK:BMAL1 mutants (Huang et al., 2014). It has been shown that neither subunit alone binds DNA well without dimerization, so mutations that decreased dimerization also decreased DNA binding.


IV. Dimerization

When forming the CLOCK:BMAL1 heterodimer complex, each domain interacts with the corresponding domain of the other subunit (For example: CLOCK bHLH with BMAL1 bHLH). One of the most important components of the interaction between the bHLH subunits in formation of the heterodimer is the hydrophobic core in the four-helical bundle, with mutation leading to a great reduction in the ability to form a stable heterodimeric complex.

Interaction between the PAS-A domains of the subunits was shown to be mainly facilitated by hydrophobic interactions. This is primarily from of the A'alpha helix of CLOCK with the beta-sheet face of BMAL1. Specific residues involved in this interaction were shown to be in CLOCK and in BMAL1, with double mutation to E and D respectively making association between the subunits undetectable (Huang et al., 2012).

On the PAS-B domains, the most involved in dimerization are the CLOCK helical face and the beta-sheet face of BMAL1. It was shown that altered PAS-B domain interactions resulted from mutations to CLOCK W284, V315 and BMAL1 V435, decreasing dimerization of the proteins. There was a significant decrease in dimerization when mutating BMAL1 , significantly altering the hydrophobic interactions. One of the main residue contacts involved in the interaction of the PAS-B domain is between . The double mutation of these residues to A led to a decrease in dimerization as well as transactivation activity (Huang et al., 2012). This confirms the unusual PAS-B domain association involving the CLOCK helical face and the BMAL1 beta-sheet face as seen in the crystal structure. 


V. Role in Circadian Cycling

CLOCK and BMAL1 are able to regulate the circadian clock in mammals through physical interaction with regulators PER and CRY. The specifics of this interaction are not known on a structural basis, but the binding of these repressors could affect DNA binding, change transactivation potential, or even reshape interactions with other activators or repressors. It has been shown that overexpression of CLOCK leads to a shorter circadian cycle while overexpression of BMAL1 either lengthens or does not affect the cycle.

Research suggests that CRY interacts with the PAS-B domain of CLOCK and with a C-terminal region of BMAL1. Explicit amino acid residues involved in the interaction of the CLOCK PAS-B domain in repression by CRY are . These residues are located on the solvent exposed HI loop of the beta-sheet face and are readily available for interaction with the repressor. CRY binding to CLOCK is also consistent with the fact that CRY is a highly positively charged protein and the exposed surfaces of CLOCK have negative charges.

Interaction of BMAL1 with the PER repressor is not as well defined. Structural similarities between the tandem PAS domains of the proteins show possible structural or functional conservation. Further, the residue located at the HI loop of the BMAL1 PAS-B domain (which corresponds to CLOCK W362, involved in association with CRY) is conserved in Drosophila and mouse PER proteins and is involved in the formation of homodimers with a second PER protein. This suggests the importance of a conserved W residue at this position mediating protein-protein interactions. 


VI. References

Huang, N., Chelliah, Y., Shan, Y., Taylor, C., Yoo, S., Partch, C., Green, C., Zhang, H., and Takahashi, J. (2012). Crystal Structure of the Heterodimeric CLOCK:BMAL1 Transcriptional Activator Complex. Science, 337(6091), pp. 189-194

Kewley, R., Whitelaw, M., and Chapman-Smith A. (2004). The Mammalian Basic helix-loop-helix/PAS Family of Transcriptional Regulators. The International Journal of Biochemistry and Cell Biology, 36(2), pp. 189-204

Menet, J., Pescatore, S., and Rosbash, M. (2014). CLOCK:BMAL1 is a Pioneer-like Transcription Factor. Genes and Development, 28:8-13.

Sahar, S. and Sassone-Corsi, P. (2009). Metabolism and Cancer: the Circadian Clock Connection. Nature Reviews Cancer, 9:886-896.

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