Sandbox Reserved 973
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| This Sandbox is Reserved from 15/11/2014, through 15/05/2015 for use in the course "Biomolecule" taught by Bruno Kieffer at the Strasbourg University. This reservation includes Sandbox Reserved 951 through Sandbox Reserved 975. |
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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 hormons level.One one the most important protein in this rhythm is the heterodimeric complex CLOCK:BMAL1, which is a transcriptionnal factor responseable for the activation of 2 type of genes; Period(Per1,Per2) and Cryptochrome(Cry1,Cry2), by interacting with the E-box DNA. In deed mutants mice devoid of CLOCK or BMAL1 proteins have seriously disrupted rhythm, at a molecular level as well as on the behavioural one. This is a central mechanism in the circadian cycle regulation as 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. On the other hand, CLOCK:BMAL 1 is also able to activate the transcription of retinoic acid-related nuclear receptors: 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 modifiers proteins can modify the stabibility or influence the translocation of some core clock actors, including BMAL1 and CLOCK. That's the case of 2 types of Casein kinase 1 (CK1). Mutations in these modifiers can shorten the circadian cycle of the mamals and cause serious sleep disorders. Nevertheless recents studies showed that every 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.
The two peptides involved in the dimere have very similar sequences. In mus musculusBMAL1 is 387 residues long and CLOCK is 361. Both of these subunits are basic helix-loop-helix-PAS proteins (bHLH-PAS) which contains the same 3 particular domains: a bHLH domain, a PAS-A domain and a PAS-B domain. 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.
Domains
Basic helix-loop-helix domains are especially composed by 2 C-terminal helices called α1 and α2 that are involved in the formation of a canonical four-helical bHLH bundle. This bond between the HLH domains helps to stabilize the heterodimeric complex as the core of the the bundle is very hydrophobic. The spatial arrangement of this assembly has a major role in the the E-box recognition. The α1 helices are responseable 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 ordre to interact with the major grive of DNA duplex.In fact, when Leu57 and Leu74 of CLOCK, and Leu95 and Leu115 of BMAL1 are mutated to glutamate, mutants show no transactivation activity anymore bécasse the ability to form stable four-helice bundle is reduced as we can observe it through a bimolecular fluorescence complementation (BiFC) assay. in addition most of these mutations tend to unsettle the full length hétérodimeric complex.
PAS-A domains don't have the same conformation in the two subunits. In BMAL1, we can observe 3 loops involving about 60 residues whereas in CLOCK there are only 25 residues in a single loop. Nevertheless, this 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 BMAL1 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 subunits) as C:L113E+B:I317D, was responseable for a 25% level. In the same time, no full length complex was detected.
in both subunits the two PAS domains are linked thanks to an ADN linker of approximately 15 residues called L2 but the conformation of this linker is very different.
In CLOCK the main part of L2 is buried between the dimeric interface whereas in BMAL1 the linker is exposed on the outside and is very flexible. The PAS-B domaines are stacked in a parallel way. The sheet of BMAL1 contacts the helical face of CLOCK so several residues get hidden on CLOCK as well as on BMAL1, 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 -sheet intrudes 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% réduction for some aminoacids. We also observe a sensible destabilization of the PAS-B domainns interactions wich enlightens the importance of its primary structure. What's more, 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 activity of the protrein. 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
Structural highlights
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