Sandbox Reserved 973
From Proteopedia
|
Contents |
Function and overall structure
The pacemaker of the circadian system is the suprachiamastic nucleus (SNC) located in the hypothalamus. This center controls the circadian rhythm through the coordination of peripheric oscillators all over the organism. The mechanisms that occur in the peripheric system are, at a molecular level, very similar to those in the SNC; that is to say a network of transcriptional and translational regulations, creating different loops that take about 24 hours to complete. This circadian cycle regulates many physiologic parameters and coordonates several phenomenon such as sleep period or hormon levels. One of the most important proteins in this rhythm is the heterodimeric complex CLOCK:BMAL1, which is a transcriptionnal factor responsible for the activation of 2 type of genes; Period(Per1,Per2) and Cryptochrome(Cry1,Cry2), by interacting with the E-box DNA. Indeed mutants mice devoid of or proteins have seriously disrupted rhythm, at a molecular level as well as on their behavior.
The two polypeptides involved in the dimere have very similar sequences. The structure of and have mostly been studied in mus musculus. Both of these subunits are basic helix-loop-helix-PAS proteins (bHLH-PAS) which contain the same 3 particular domains: a domain, a PAS-A domain and a PAS-B domain, plus a transactivator domain in the C-terminal region. They are involved in DNA binding and dimerization abilities. Mutations that affects the heterodimer interfaces can then disturb the activity of the complex and therefore the persistence and periodicity of the circadian cycle. Indeed the two subunits are tightly intertwined as each domain of interact with the corresponding one of . Another important feature of this heterodimere is that there is an assymetric distribution of the electrostatic potential. tends to have a global negative charge while has a positive one. The fact that the two subunits of the complex expose this charged surface in the 3D structure match with the hypothesis that and are not involved in the same interaction with the other regulatory proteins listed before.
Domains of the subunits
This part deals with the structure of the 3 domains involved in the dimerization pattern. The transactivator domains of the two subunits, which are both transcription factors, working as positive elements in the circadian molecular clock but they will not be presented here. The data exposed below were obtained by crystallographic analysis with mouse (residues 26–384) and (residues 62–447)[1].
bHLH domain
domains of and are especially composed by 2 C-terminal helices called α1 and α2. These two helices are involved in the formation of a canonical four-helical bHLH bundle. Given the fact that the core of the bundle is very hydrophobic, the bond between the domains helps to stabilize the heterodimeric complex. The spatial arrangement of this assembly has a major role in the E-box recognition. The α1 helices are responsible for the DNA binding and the aminoacids sequence is crucial. Site-directed mutagenesis experiments showed that some hydrophobic residues, leucine in particular, were necessary in order to interact with the major groove of DNA duplex. In fact, when Leu57 and Leu74 of CLOCK, and Leu95 and Leu115 of are mutated to glutamate, mutants show no transactivation activity anymore because the ability to form stable four-helices bundle is reduced. We can observe it through a bimolecular fluorescence complementation (BiFC) assay. In addition, most of these mutations tend to unsettle the full length of the heterodimeric complex.
PAS-A domain
PAS-A domains don't have the same conformation in the two subunits. In , we can observe 3 loops involving about 60 residues whereas in CLOCK there are only 25 residues in a single loop [2]. Nevertheless, these two PAS-A domains adopt a typical PAS fold. The core of these domains contains a five-stranded antiparallel β-sheet (AβBβGβHβIβ) as well as numerous α helices (Cα, DαEαFα). They also contain an N-terminal A'α helix that does not belong to the canonical PAS fold. Those helices pack in between the β-sheet faces and are involved in the dimerization interactions. The two PAS-A domains are mostly linked thanks to hydrophobic bonds. Indeed, Phe104, Leu105, and Leu113 on the A′α helix of CLOCK are interacting with the residues Leu159 on strand Aβ, Thr285 and Tyr287 on Hβ, Val315 and Ile317 on strand Iβ, of the subunit. The same kind of bonds are occuring between the A'α helix of BMAL1 and the β-sheet of CLOCK. Thus, the two PAS-A domains form a parallel dimer. Once more the right position of key aminoacids is necessary to the dimerization process. BMAL1 mutant I317D see their transactivation decreased to 80% of the control population and a double mutation (one on each subunit) as C:L113E+B:I317D, was responsible for a 25% level. In the same time, no full length complex was detected.
PAS B domain
In both subunits, the two PAS domains are linked thanks to an ADN linker called L2. L2 consists of approximately 15 residues, but the conformation of this linker is very different in the subunits. In CLOCK the main part of L2 is buried between the dimeric interface, whereas in the linker is exposed on the outside and is very flexible. The PAS-B domains are stacked in a parallel way. The of BMAL1 contacts the helical face of CLOCK, so several residues get hidden on CLOCK as well as on , including Tyr310, Val315, Leu318 of the first one and Phe423, Trp427 and Val435 of the second one. Hydrophobic interactions are once more involved in the dimerization process. As an exemple, BMAL1 Trp427 located in the inserts itself in a hydrophobic cleft created by the CLOCK helical face fold, where it contacts the indole ring of CLOCK Trp248. Single mutations on the two PAS-B domain seem to have very limited effects on the activity, even if it can raise to a 30% reduction for some aminoacids. We also observe a sensible destabilization of the PAS-B domains interactions, which enlightens the importance of its primary structure. Moreover, the double BMAL1 PAS-B domain mutant, B:F423R/V435R and the combined CLOCK:BMAL1 mutant C:W284A+B:W427A showed a decrease of the heterodomeric complex concentration and of the protein activity. This result points out the importance of the contact between CLOCK Trp 248 and BMAL1 Trp 427 as explained previously.
Effects on the circadian cycle
Interaction with Per and Cry genes, but also PER and CRY proteins is a central mechanism in the circadian cycle regulation. The downstream products PER and CRY can then accumulate and dimerize too, so they can repress transcription of Bmal1 and Clock at night, after they relocated in the cell nucleus, creating an autoregulatory feedback loop. These interactions are not completly elucitaded yet but some cristallography and site-directed mutagenesis experiments brought some new elements. CRY and PER seem to interact with CLOCK:BMAL1 to form a bigger repression complex but the mechanism of the repression activity is still unknown. CRY proteins are likely to bind on the CLOCK PAS-B domain thanks to the presence of the β-sheet or on the C-terminal region of BMAL1. The global positive charge of CRY also suggests an interaction with the CLOCK PAS domains as it is negatively charged. Mutations of residues Gln332, His360, Gln361, Trp362 and Glu367 of the CLOCK PAS-B domain delay the repression by CRY, which is another argument in favor of this hypothesis. What's more an unexpected similary was found between the BMAL1 PAS domains and the PER proteins PAS domains. This suggests an interaction of PER with through this interface. One of the clues is the presence of a tryptophane residue which is very conserved in the 2 types of polypeptides. The BMAL1 Trp427 is also present in PER where it mediates the formation of an homodimer. It may be a key residue for the binding of PER.
On the other hand, CLOCK:BMAL 1 is also able to activate the transcription of retinoic acid-related nuclear receptors[3]: Rev-erbα and Rorα.These proteins have the ability to regulate the transcription of Bmal1, RORα as an activator and REV-ERBα as a repressor. But this molecular clock is also regulated by post-translational modifications of the subunits, notably phosphorylation and ubiquitination. Some modifier proteins can change the stabibility or influence the translocation of some core clock actors, including and . That is the case of 2 types of Casein kinase 1 (CK1). Mutations in these modifiers can shorten the circadian cycle of the mammals and cause serious sleep disorders.
Mutagenesis experiments showed that is normally in excess of , which imply that is the limiting factor for the dimerization. Indeed, an overexpression of Clock will make the cycle shorter, whereas an overexpression of Bmal1 may have no effect on the cycle length or make it a little longer. Nevertheless, recent studies showed that each cell has its autonomy towards the circadian cycle and it is important to notice that the two genes Clock and Bmal1 are not identically expressed in every tissues, and that the two proteins don't act the same way in this different cell types. Clock mRNA follows a transcriptional cycle in the peripheric oscillators whereas it is constitutively expressed in the SCN. Futhermore the different ROR proteins which have feedback effects on Bmal1 transcription are expressed in a tissue-specific manner, so has various functions at various moments. Again, doesn't have the same influence on downstream regulated genes, depending on the tissue. For example, CLOCK has a limited impact on the amplitudes of the Rev-erbα mRNA oscillation in the SCN. But Clock−/− mice show a important decrease of the same amplitude in the liver. Thus, it is important to keep in mind that the CLOCK:BMAL1 complex subunits are expressed in tissue-specific patterns and that the heterodimer makes a tissue-specific regulation of the circadian clock, which drive the study of the complex functions very difficult.
Diseases
As we mentionned it above, several mutations of the complex can alter the circadian rhythm. These mutations can affect the structure of the heterodimer and its ability to bind DNA, endanger its stability or modify the interactions with co-regulator clock elements. However, many of them are responsible for numerous pathologies as they have an impact on the whole clock system, involved in different functions, depending on the tissue type. In mammals, an increasing number of disorders are thought to be linked with the clock proteins dysfunction. For the single CLOCK:BMAL1 complex, mutations are related to various troubles such as infertility, progressive arthropathy, abnormal gluconeogenesis, abnormal lipogenesis or altered sleep pattern. The complex plays a major role in cellular metabolic pathway, so it is linked to hepatic steatosis, hyperleptinemia, hyperglycemia and hypoinsulinemia. is also necessary for embryonic fibroblast cells to differenciate into adipocytes. Another unexpected aspect of the heterodimer functions is that it can influence the efficacy of some drugs. For example, muatnts that don't own this complex show a good response to chemotherapy all day long whereas it varies for the wild mice. Moreover, other disrupted genes such as mutated Per or Cry must be taken into account because they also disturb the proper working of the complex, leading to other disorders.
Structural highlights
This is a sample scene created with SAT to by Group, and another to make of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.
</StructureSection>
References
- ↑ Crystal Structure of the Heterodimeric CLOCK:BMAL1 Transcriptional Activator Complex by Huang, N., Chelliah, Y., Shan, Y., Taylor, C.A., Yoo, S.H., Partch, C., Green, C.B., Zhang, H., Takahashi, J.S DOI: 10.1126/science.1222804
- ↑ Crystal Structure of the Heterodimeric CLOCK:BMAL1 Transcriptional Activator Complex by Nian Huang, Yogarany Chelliah, Yongli Shan, Clinton A. Taylor, Seung-Hee Yoo, Carrie Partch, Carla B. Green, Hong Zhang, and Joseph S. Takahashi doi: 10.1126/science.1222804
- ↑ Molecular components of the mammalian circadian clock by Caroline H. Ko and Joseph S. Takahashi
