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This is a default text for your page '. Click above on edit this page' to modify. Be careful with the < and > signs. You may include any references to papers as in: the use of JSmol in Proteopedia ^[1] or to the article describing Jmol ^[2] to the rescue.

Function and overall structure

The heterodimeric complex CLOCK:BMAL1 is a transcriptionnal factor responseable for the activation of 2 genes; Period(Per1,Per2) and Cryptochrome(Cry1,Cry2), by interacting with the E-box DNA. This is a central mechanism in the circadian cycle regulation as the downstream products PER and CRY can accumulate dimerize too, so they can rpress transcription of Bmal1 and Clock at night, creating an autregulatory feedback loop. The two peptides involved in the dimere have very similar sequences. Chez mus musculusBMAL1 is 387 residues long when CLOCK is 361. Both of these subunits are basic helix-loop-helix-PAS proteins (bHLH-PAS) which contains the same 3 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

bHLH domains are especially composed by 2 C-terminal helices called 1 and 2 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 1 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 2 PAS-A domains form a parallel dimer. Once more the 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, were 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 il contacts the indole ring of CLOCK Trp248.

Relevance

Structural highlights

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References

1. ↑ Hanson, R. M., Prilusky, J., Renjian, Z., Nakane, T. and Sussman, J. L. (2013), JSmol and the Next-Generation Web-Based Representation of 3D Molecular Structure as Applied to Proteopedia. Isr. J. Chem., 53:207-216. doi:http://dx.doi.org/10.1002/ijch.201300024
2. ↑ Herraez A. Biomolecules in the computer: Jmol to the rescue. Biochem Mol Biol Educ. 2006 Jul;34(4):255-61. doi: 10.1002/bmb.2006.494034042644. PMID:21638687 doi:10.1002/bmb.2006.494034042644