Sandbox Reserved 973: Difference between revisions

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=== 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 <scene name='60/604492/Bmal1/1'>BMAL1</scene> the linker is exposed on the outside and is very flexible. The PAS-B domains are stacked in a parallel way. The β-sheet of BMAL1 contacts the helical face of CLOCK, so several residues get hidden on CLOCK as well as on <scene name='60/604492/Bmal1/1'>BMAL1</scene>, 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 β-sheet inserts itself in a hydrophobic cleft created by the CLOCK helical face fold, where it contacts the indole ring of CLOCK Trp248.

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 <scene name='60/604492/Bmal1/1'>BMAL1</scene> the linker is exposed on the outside and is very flexible. The PAS-B domains are stacked in a parallel way. The <scene name='60/604492/Beta_sheet/1'>β-sheet</scene> of BMAL1 contacts the helical face of CLOCK, so several residues get hidden on CLOCK as well as on <scene name='60/604492/Bmal1/1'>BMAL1</scene>, 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 <scene name='60/604492/Beta_sheet/1'>β-sheet</scene> 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.

Sandbox Reserved 973: Difference between revisions

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@@ Line 27: / Line 27: @@
 === 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 <scene name='60/604492/Bmal1/1'>BMAL1</scene> the linker is exposed on the outside and is very flexible. The PAS-B domains are stacked in a parallel way. The β-sheet of BMAL1 contacts the helical face of CLOCK, so several residues get hidden on CLOCK as well as on <scene name='60/604492/Bmal1/1'>BMAL1</scene>, 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 β-sheet inserts itself in a hydrophobic cleft created by the CLOCK helical face fold, where it contacts the indole ring of CLOCK Trp248.
+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 <scene name='60/604492/Bmal1/1'>BMAL1</scene> the linker is exposed on the outside and is very flexible. The PAS-B domains are stacked in a parallel way. The <scene name='60/604492/Beta_sheet/1'>β-sheet</scene> of BMAL1 contacts the helical face of CLOCK, so several residues get hidden on CLOCK as well as on <scene name='60/604492/Bmal1/1'>BMAL1</scene>, 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 <scene name='60/604492/Beta_sheet/1'>β-sheet</scene> 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.