Sandbox Reserved 973: Difference between revisions

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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 <scene name='60/604492/Clock/1'>CLOCK</scene> or <scene name='60/604492/Bmal1/1'>BMAL1</scene> 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 <scene name='60/604492/Clock/1'>CLOCK</scene> and <scene name='60/604492/Bmal1/1'>BMAL1</scene> 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 ~~bHLH~~ 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 <scene name='60/604492/Clock/1'>CLOCK</scene> interact with the corresponding one of <scene name='60/604492/Bmal1/1'>BMAL1</scene>.

The two polypeptides involved in the dimere have very similar sequences. The structure of <scene name='60/604492/Clock/1'>CLOCK</scene> and <scene name='60/604492/Bmal1/1'>BMAL1</scene> 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 <scene name='60/604492/Bhlh/1'>TextToBeDisplayed</scene> 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 <scene name='60/604492/Clock/1'>CLOCK</scene> interact with the corresponding one of <scene name='60/604492/Bmal1/1'>BMAL1</scene>.

Another important feature of this heterodimere is that there is an assymetric distribution of the electrostatic potential. <scene name='60/604492/Clock/1'>CLOCK</scene> tends to have a global negative charge while <scene name='60/604492/Bmal1/1'>BMAL1</scene> 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 <scene name='60/604492/Clock/1'>CLOCK</scene> and <scene name='60/604492/Bmal1/1'>BMAL1</scene> are not involved in the same interaction with the other regulatory proteins listed before.

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=== bHLH domain ===

Basic helix-loop-helix domains of CLOCK and <scene name='60/604492/Bmal1/1'>BMAL1</scene> 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 ~~bHLH~~ 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 <scene name='60/604492/Bmal1/1'>BMAL1</scene> 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.

<scene name='60/604492/Bhlh/1'>Basic helix-loop-helix</scene> domains of <scene name='60/604492/Clock/1'>CLOCK</scene> and <scene name='60/604492/Bmal1/1'>BMAL1</scene> 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 <scene name='60/604492/Bhlh/1'>TextToBeDisplayed</scene> 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 <scene name='60/604492/Bmal1/1'>BMAL1</scene> 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 <scene name='60/604492/Bmal1/1'>BMAL1</scene>, we can observe 3 loops involving about 60 residues whereas in CLOCK there are only 25 residues in a single loop. 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.

PAS-A domains don't have the same conformation in the two subunits. In <scene name='60/604492/Bmal1/1'>BMAL1</scene>, we can observe 3 loops involving about 60 residues whereas in CLOCK there are only 25 residues in a single loop <ref>10.1126/science.1222804</ref>. 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 <scene name='60/604492/Bmal1/1'>BMAL1</scene> 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.

Sandbox Reserved 973: Difference between revisions

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@@ Line 6: / Line 6: @@
 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 <scene name='60/604492/Clock/1'>CLOCK</scene> or <scene name='60/604492/Bmal1/1'>BMAL1</scene> 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 <scene name='60/604492/Clock/1'>CLOCK</scene> and <scene name='60/604492/Bmal1/1'>BMAL1</scene> 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 bHLH 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 <scene name='60/604492/Clock/1'>CLOCK</scene> interact with the corresponding one of <scene name='60/604492/Bmal1/1'>BMAL1</scene>.
+The two polypeptides involved in the dimere have very similar sequences. The structure of <scene name='60/604492/Clock/1'>CLOCK</scene> and <scene name='60/604492/Bmal1/1'>BMAL1</scene> 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 <scene name='60/604492/Bhlh/1'>TextToBeDisplayed</scene> 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 <scene name='60/604492/Clock/1'>CLOCK</scene> interact with the corresponding one of <scene name='60/604492/Bmal1/1'>BMAL1</scene>.
 Another important feature of this heterodimere is that there is an assymetric distribution of the electrostatic potential. <scene name='60/604492/Clock/1'>CLOCK</scene> tends to have a global negative charge while <scene name='60/604492/Bmal1/1'>BMAL1</scene> 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 <scene name='60/604492/Clock/1'>CLOCK</scene> and <scene name='60/604492/Bmal1/1'>BMAL1</scene> are not involved in the same interaction with the other regulatory proteins listed before.
@@ Line 16: / Line 16: @@
 === bHLH domain ===
-Basic helix-loop-helix domains of CLOCK and <scene name='60/604492/Bmal1/1'>BMAL1</scene> 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 bHLH 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 <scene name='60/604492/Bmal1/1'>BMAL1</scene> 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.
+<scene name='60/604492/Bhlh/1'>Basic helix-loop-helix</scene> domains of <scene name='60/604492/Clock/1'>CLOCK</scene> and <scene name='60/604492/Bmal1/1'>BMAL1</scene> 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 <scene name='60/604492/Bhlh/1'>TextToBeDisplayed</scene> 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 <scene name='60/604492/Bmal1/1'>BMAL1</scene> 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 <scene name='60/604492/Bmal1/1'>BMAL1</scene>, we can observe 3 loops involving about 60 residues whereas in CLOCK there are only 25 residues in a single loop. 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.
+PAS-A domains don't have the same conformation in the two subunits. In <scene name='60/604492/Bmal1/1'>BMAL1</scene>, we can observe 3 loops involving about 60 residues whereas in CLOCK there are only 25 residues in a single loop <ref>10.1126/science.1222804</ref>. 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 <scene name='60/604492/Bmal1/1'>BMAL1</scene> 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.