Sandbox Reserved 973: Difference between revisions
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== Domains == | == Domains of the subunits == | ||
=== | === bHLH domain=== | ||
Basic helix-loop-helix domains of CLOCK and BMAL1 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 domain == | |||
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. | 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. | ||
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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. | 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. | ||
=== PAS B domain === | |||
In both subunits the two PAS domains are linked thanks to an ADN linker called L2 of approximately 15 residues 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. | |||
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. | 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== | == Effects on the circadian cycle== | ||
== Diseases == | |||
== Structural highlights == | == Structural highlights == | ||
