Proteopedia

User:Alice Harmon/Sandbox 4: Difference between revisions

← Older edit
No edit summary
No edit summary
 
(13 intermediate revisions by the same user not shown)
Line 4: Line 4:
[[Image:ABA model 2 med.jpg|left]]
[[Image:ABA model 2 med.jpg|left]]
==Role in Abscisic Acid signaling==
==Role in Abscisic Acid signaling==
SnRK2.6/OST1/SRK2E is a triply-named protein kinase from Arabidopsis, which is activated by the abscisic acid (ABA) response pathway in plants<ref name = "Mustilli2002">PMID:12468729</ref><ref>PMID:12514244</ref><ref name = "Nakashima2009">PMID:19541597</ref><ref name = "Fujii2007">PMID:17307925</ref>. As shown in the figure, in unstimulated cells SnRK2.6/OST1/SRK2E (K in the figure) and a protein phosphatase 2C (ABI1 or its homologs; P in the figure) are in a complex (K-P) in which the kinase is dephosphorylated and inactive<ref name ="Umezawa2009"> PMID:19805022 </ref><ref name ="Soon2013"> PMID:22116026 </ref>. The kinase and phosphatase are proposed to be tethered via the C-terminal sequence (dashed line) of the kinase<ref name ="Umezawa2009"/><ref name ="Soon2013"/><ref>PMID:16365038</ref>. When ABA levels in the cytosol rise,  ABA binds to an ABA receptor (PYR1/PYL/RCAR; R in the figure)<ref>PMID:19893533</ref><ref>PMID:19407143</ref><ref>PMID:19407142</ref>. The activated receptor (R<sup>.</sup>ABA) binds to the protein phosphatase (R<sup>.</sup>ABA-P) and inactivates it. SnRK2.6/OST1/SRK2E is still tethered to the phosphatase, but its active site is now free to be activated by autophosphorylation or phoshorylation by another protein kinase <ref>PMID:17103012</ref><ref>PMID:16980311</ref>.  Activation of SnRK2.6/OST1/SRK2E leads to phosphorylation of: 1) ion channels SLAC1<ref>PMID: 19955405</ref> and KAT1<ref>PMID: 19785574</ref> in guard cells and stomatal closure; 2)  transcription factor ABI5<ref name = "Nakashima2009"/> in seeds/seedlings and dormancy/growth arrest; or 3) phosphorylation of transcription factor AREB/ABF <ref>PMID: 16446457</ref><ref name = "Fujii2007"/> in vegetative tissue and stress tolerance and growth regulation.  
SnRK2.6/OST1/SRK2E is a triply-named protein kinase from Arabidopsis, which is activated by the abscisic acid (ABA) response pathway<ref name = "Mustilli2002">PMID:12468729</ref><ref>PMID:12514244</ref><ref name = "Nakashima2009">PMID:19541597</ref><ref name = "Fujii2007">PMID:17307925</ref>. As shown in the figure, in unstimulated cells SnRK2.6/OST1/SRK2E (K) and a protein phosphatase 2C (P) are in a complex (K-P) in which the kinase is dephosphorylated and inactive<ref name ="Umezawa2009"> PMID:19805022 </ref><ref name ="Soon2012"> PMID:22116026 </ref>. The kinase and phosphatase are proposed to be tethered via the C-terminal sequence (dashed line) of the kinase<ref name ="Umezawa2009"/><ref name ="Soon2012"/><ref>PMID:16365038</ref>. When ABA levels in the cytosol rise,  ABA binds to an ABA receptor (R)<ref>PMID:19893533</ref><ref>PMID:19407143</ref><ref>PMID:19407142</ref>. The activated receptor (R<sup>.</sup>ABA) binds to the protein phosphatase (R<sup>.</sup>ABA-P) and inactivates it. SnRK2.6/OST1/SRK2E, although still tethered to the phosphatase, is now free to be activated by autophosphorylation or phoshorylation by another protein kinase<ref>PMID:17103012</ref><ref>PMID:16980311</ref>.  Activation of SnRK2.6/OST1/SRK2E leads to phosphorylation of: 1) ion channels SLAC1<ref>PMID: 19955405</ref> and KAT1<ref>PMID: 19785574</ref> in guard cells and stomatal closure; 2)  transcription factor ABI5<ref name = "Nakashima2009"/> in seeds/seedlings and dormancy/growth arrest; or 3) phosphorylation of transcription factor AREB/ABF <ref>PMID: 16446457</ref><ref name = "Fujii2007"/> in vegetative tissue and stress tolerance and growth regulation.  


Structures in the figure are: R, apo pYR2, [[3kdh]];  R<sup>.</sup>ABA, PYR2<sup>.</sup>ABA, [[3kdi]]; K-P; SnRK2.6-HAB1, [[3ujg]]; R<sup>.</sup>ABA-P, PYR2<sup>.</sup>ABA-ABI2, [[3ujl]]; K, SnRK2.6,[[3uc4]].
Structures in the figure are: R, apo pYR2, [[3kdh]];  R<sup>.</sup>ABA, PYR2<sup>.</sup>ABA, [[3kdi]]; K-P; SnRK2.6-HAB1, [[3ujg]]; R<sup>.</sup>ABA-P, PYR2<sup>.</sup>ABA-ABI2, [[3ujl]]; K, SnRK2.6,[[3uc4]].


==Kinase names and family members==
==Kinase names and family members==
Line 16: Line 15:
==Kinase structure and regulation==
==Kinase structure and regulation==


SnRK2.6/OST1/SRK2E has a primary structure comprising an amino terminal [[Eukaryotic Protein Kinase Catalytic Domain]] and a C-terminal sequence that contains the SNRK2 box, which is unique to the SNRK2 family and required for activity<ref name ="Belin2006"> PMID: 16766677</ref>. Its C-terminus also contains a sequence called the ABA box, which is found only in the family members that are responsive to ABA<ref name ="Belin2006">. The latter sequence is required for binding to PP2C<ref name ="Belin2006">, but is not seen in the crystal structure (why??) (see below).
SnRK2.6/OST1/SRK2E has a primary structure comprising an amino terminal [[Eukaryotic Protein Kinase Catalytic Domain]] and a C-terminal sequence that contains the SNRK2 box, which is unique to the SNRK2 family and required for activity<ref name= "Ng2011"/><ref name ="Belin2006"> PMID: 16766677</ref>. Its C-terminus also contains a sequence called the ABA box, which is found only in the family members that are responsive to ABA<ref name ="Belin2006">. The latter sequence is required for binding to PP2C<ref name ="Belin2006">, but is not seen in the crystal structure.


<ref name ="Soon2013"> PMID:22116026 </ref>
<ref name ="Soon2013"> PMID:22116026 </ref>


{|
{|
|'''Left scene''' - The crystal structure [[3uc4]] shows that the catalytic domain of SnRK2.6 is typical of [[Eukaryotic Protein Kinase Catalytic Domain]]) except for an additional α-helix in the small lobe, which is formed by SNRK2 box sequence. This helix parallels subdomain III and resembles **blah blah** kinase structures.
|'''Left scene''' - unphosphorylated SnRK2.6 without any ligands [[3uc4]]<ref name = "Ng2011">PMID:22160701</ref>
<br>
|'''Right scene''' - SnRK2.6 (blue) in complex with the protein phosphatase 2C, HAB1 (gold), with Mg<sup>2+</sup> and SO<sub>4</sub><sup>2-</sup> [[3ujg]]<ref name = "Soon2012"/>
|'''Right scene''' - The crystal structure [[3ujg]] shows the SnRK2.6–HAB1 complex, which was constructed as a fusion protein with a H6-tag (MAHHHHHHA) at the N-terminus of SnRK2.6 (residues 11–362) fused to HAB1(172–511) with a GSGSAGSAAGS linker. Residues 319-362 of SnRK2.6, which includes the ABA box, and the linker sequence are not resolved in the 3D structure.  D296A and E297A are mutations introduced at the crystal packing interface to reduce surface entropy.
|-
|-
| <applet load='3ujg' size='400' frame='true' align='left' caption='3uc4 - Apo SnRK2.6' scene = '55/559985/Aposnrk2_6/3' /><br  clear='both'>'''3uc4 scenes''' <Br><scene name='55/559985/Aposnrk2_6/3'>1. Default scene</scene><Br><scene name='55/559985/Aposnrk2_6critical/1'>2. Important structures</scene>Activation loop (with gap) in blue<br>
| <applet load='3ujg' size='400' frame='true' align='left' caption='3uc4 - Apo SnRK2.6' scene = '55/559985/Aposnrk2_6/6' /><br  clear='both'>'''3uc4 scenes''' <Br><scene name='55/559985/Aposnrk2_6/7'>1. Default scene</scene> The catalytic domain of SnRK2.6 is typical of [[Eukaryotic Protein Kinase Catalytic Domain]]) except for an additional α-helix (shown as strands) in the small lobe, which is formed by SNRK2 box sequence.  <Br><scene name='55/559985/Aposnrk2_6critical/3'>2. Important structures:</scene> The activation segment (with unresolved gap), including the D of the DFG motif in ball and stick, is blue. The catalytic loop, including the D of the DLKLEN motif in ball and stick, is orchid. Subdomain III, including its invariant E in ball and stick, is gold. The invariant K of subdomain II is in chartreuse. The SnRK2 box is turquoise. The C-terminal domain, that includes the ABA box is unresolved. The arrangement of the residues in ball and stick around the active site, indicate that this structure is in a partially active state in spite of its unphosphorylated activation loop. This is possibly due to the interaction of the SNRK2 box helix with subdomain III.<ref name = "Ng2011"/>. The interaction between these helices is similar to the interaction of helices in the complex between <scene name='55/559985/Cdk2-cyclin/3'>cyclin-dependent protein kinase 2 (CDK2) and cyclin</scene> [[1w98]].  Here we see that subdomain III of the protein kinase (opaque blue) is stablized by interaction with a helix from cyclin (opaque gold). The positioning of subdomain III by this interaction is critical for for formation of the active site.<ref>PMID:15660127</ref>. <br><br><br><br><br><br><br><br><br><br>
Catalytic loop in orchid<br>
| <applet load='3ujg' size='400' frame='true' align='left' caption='3ujg - SnRK2.6-HAB1' scene = '55/559985/Aposnrk2_6/2' /><Br clear='both'>'''3ujg scenes'''<Br><scene name='55/559985/Aposnrk2_6/2'>1. Default Scene</scene> The two enzymes are bound via interface their active sites. The phosphatase inactivates the kinase by dephosphorylating the kinase activation loop and by sterically blocking the kinase active site. The complex was constructed as a fusion protein with a 6His-tag at the N-terminus of SnRK2.6 (residues 11–362) fused to HAB1(172–511) via a GSGSAGSAAGS linker. Mutations of D296A and E297A in SnRK2.6 were introduced at the crystal packing interface to reduce surface entropy. <br><scene name='55/559985/Ost1hab1_critical/3'>2. Important structures in SnRK2.6</scene> The same structures as in the left scene are shown. The fully resolved activation segment extends into the phosphatase's active site and is unphosphorylated. Residues 319-362 of SnRK2.6, which includes the ABA box, and the GSGSAGSAAGS linker are not resolved. The disorganization of the residues shown in ball and stick, with most pointing away from the active site, indicates that the catalytic domain is in the inactive state.<Br><scene name='55/559985/Ost1hab1_interaction/2'>3. Zone of interaction</scene> The activation loop (blue trace) of SnRK2.6 is inserted into the catalytic site (marked by the magnesium ions) of the phosphatase. The phosphorylatable residue of the activation loop S175 (CPK ball and stick) is positioned near the magnesium ions. W385 of the phosphatase (brown ball and stick) in turn protrudes into the kinase's active site, where it interacts with residues R139 and Glu144 (CPK ball and stick) of the catalytic loop (orchid trace) and I183 of the activation loop.<br><scene name='55/559985/Tetherbinding/1'> 5. Proposed interaction zone </scene> SnRK2.6 is shown in blue cartoon, and HAB1 in gold spacefill. The ABA box sequence is not resolved, but it would extend from the C-terminal end of the SNRK2 box helix (cyan helix). It is proposed that the ABA box sequence, which is highly acidic, binds to a patch of basic residues (blue) on the surface of the phosphatase.<ref name= "Soon2012"/>
C-helix in yellow<br>
|}
K in chartreuse<br>
Snrk2 box (unique to Snrk's) in turquoise.<br>
C-terminal acidic domain missing.<br><br><br>
| <applet load='3ujg' size='400' frame='true' align='left' caption='3hx4 - active TgCDPK1' scene = '55/559985/Aposnrk2_6/2' /><Br clear='both'>'''3ujg scenes'''<Br><scene name='55/559985/Aposnrk2_6/2'>1. Default Scene</scene> <Br><scene name='55/559985/Ost1hab1_interaction/1'>2. Zone of interaction </scene><br><scene name='55/559985/Ost1hab1_critical/3'>2. Important structures</scene>
kinase activation loop in blue<br>
phosphatase W385 in brown
|}
 
 
 
 
 
 
ABA box is not seen in the crystal structure.  


Clearest picture of Pyr1 gate - latch is in Annu Reviews article
unused: <scene name='55/559985/Aposnrk2_6/4'>Cysteines in transparent kinase</scene><br>
<scene name='55/559985/Aposnrk2_6/5'>Cysteines in space fill kinase</scene><br>
<scene name='55/559985/Ost1hab1/1'>Cysteines in the complex</scene>




Line 65: Line 42:




==References==


===PP2C structures===


[[1a6q]] – hPP2C <br />
<references/>
[[2iq1]] – hPP2C κ <br />
[[2p8e]] – hPP2C β<br />
[[2cm1]] – PP2C + Mn – Micobacterium tuberculosis<br />
[[3d8k]] – PP2C – Toxoplasma gondii<br />
[[3jrq]], [[3kdj]], [[3nmn]] – AtPP2C + Pyl1 – Arabidopsis thaliana<br />
[[3nmt]], [[3kb3]], [[3nmv]], [[3ujl]] – AtPP2C + Pyl2<br />
[[4ds8]] – AtPP2C + Pyl3 + Mn<br />
[[3rt0]] – AtPP2C (mutant) + Pyl10<br />
[[3qn1]], [[3zvu]] – AtPP2C + Pyr1<br />
[[3ujg]] – AtPP2C + SNRK2<br />
[[3ujk]] – AtPP2C <br />


 
==See Also==
==References==
[http://en.wikipedia.org/wiki/Abscisic_acid] Abscisic Acid in Wikipedia
<references/>
Retrieved from "https://proteopedia.openfox.io/w/User:Alice_Harmon/Sandbox_4"