Sandbox 208: Difference between revisions
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*'''An essential step: Rab prenylation''' | *'''An essential step: Rab prenylation''' | ||
All Rabs undergo [http://http://en.wikipedia.org/wiki/Posttranslational_modification posttranslational | All Rabs undergo [http://http://en.wikipedia.org/wiki/Posttranslational_modification posttranslational modifications] by prenylation. Prenylation is essential for Rab function and membrane association but also for its binding to GDI. | ||
In most cases, this post-translationally modification consists in the attachment of geranylgeranyl lipid groups on two C-terminus cysteines. [http://http://en.wikipedia.org/wiki/Geranylgeranylation Geranylgeranylation] is catalyzed by the [http://http://en.wikipedia.org/wiki/Prenyltransferase prenyltransferase], geranylgeranyltransferase type II (GGTrII), a heterodimer containing α- and β-subunits. GGTrII requires the activity of an accessory protein, Rab escort protein ([http://http://en.wikipedia.org/wiki/Rab_escort_protein REP]). REP is able to bind newly synthesized Rabs and to mediate their delivery to GGTrII. | In most cases, this post-translationally modification consists in the attachment of geranylgeranyl lipid groups on two C-terminus cysteines. [http://http://en.wikipedia.org/wiki/Geranylgeranylation Geranylgeranylation] is catalyzed by the [http://http://en.wikipedia.org/wiki/Prenyltransferase prenyltransferase], geranylgeranyltransferase type II (GGTrII), a heterodimer containing α- and β-subunits. GGTrII requires the activity of an accessory protein, Rab escort protein ([http://http://en.wikipedia.org/wiki/Rab_escort_protein REP]). REP is able to bind newly synthesized Rabs and to mediate their delivery to GGTrII. | ||
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== Substrate binding == | == Substrate binding == | ||
Contacts between prenylated Rab proteins and GDI are established through a combination of polar and hydrophobic interactions. These interactions involve the switch I and switch II regions | Contacts between prenylated Rab proteins and GDI are established through a combination of polar and hydrophobic interactions. These interactions involve the switch I and switch II regions of Rab and its C-terminus region, including the geranylgeranyl moiety and different regions of the domain I and II of GDI. Several conformational changes in the GDI molecule occur upon Rab binding. | ||
GDI binds to the Rab protein thanks to three interaction sites: | GDI binds to the Rab protein thanks to three interaction sites: | ||
*'''GDI-Rab Binding Platform (RBP)''', with β strands e1 and e3 and helix C, which form a separate binding site. This platform is located in domain I and interacts with the globular part of the Rab molecule. The RabGDI binding epitope contains a great number of conserved residues | *'''GDI-Rab Binding Platform (RBP)''', with β strands e1 and e3 and helix C, which form a separate binding site. This platform is located in domain I and interacts with the globular part of the Rab molecule. The RabGDI binding epitope contains a great number of conserved residues: Ile41, Gly42, Asp/Glu44 and Phe45 from Switch I; Trp62, Asp63, Ala65, Gln67, Phe/Tyr70, Thr/Ala72, Thr74, Ser/Thr75, Ser/Ala76 and Arg79 from Switch II. More precisely, the RBP is an essential structural element which form a great number of interactions with the C-terminus and Switch I of Rab. Three additional invariable residues are located on RBP, and form hydrogen bonds with the switch I region and the C-terminus of Rab. | ||
*'''GDI C-terminus Coordinating Region (CCR) or C-terminus Binding Region (CBR)'''. The CBR represents a hydrophobic cavity on the GDI surface. In fact, this region is located in the cleft between domain I and domain II. It is formed by <scene name='Sandbox_208/Residues_93-112_domain_i/2'>residues 93-112</scene> from domain I and <scene name='Sandbox_208/Residues_226-235_domain_ii/2'>residues 226-235</scene> from domain II. The CBR coordinates the flexible extended C-terminus of Rab. In most cases, the CBR is occupied by side chains of hydrophobic residues of the Rab C-terminus tail. Hydrophobic contacts between GDI and Rab are supported by a hydrogen bond involving main chain atoms. Rab primary structure analysis revealed the presence of a Rab C-terminus characteristic sequence (AXA box), with two conserved aliphatic amino acid residues (Val191 and Leu193). Mutations of one of these residues induce a decrease of Rab affinity to GDI. Therefore, the AXA box contributes to increase Rab binding affinity to GDI upon complex formation. | *'''GDI C-terminus Coordinating Region (CCR) or C-terminus Binding Region (CBR)'''. The CBR represents a hydrophobic cavity on the GDI surface. In fact, this region is located in the cleft between domain I and domain II. It is formed by <scene name='Sandbox_208/Residues_93-112_domain_i/2'>residues 93-112</scene> from domain I and <scene name='Sandbox_208/Residues_226-235_domain_ii/2'>residues 226-235</scene> from domain II. The CBR coordinates the flexible extended C-terminus of Rab. In most cases, the CBR is occupied by side chains of hydrophobic residues of the Rab C-terminus tail. Hydrophobic contacts between GDI and Rab are supported by a hydrogen bond involving main chain atoms. Rab primary structure analysis revealed the presence of a Rab C-terminus characteristic sequence (AXA box), with two conserved aliphatic amino acid residues (Val191 and Leu193). Mutations of one of these residues induce a decrease of Rab affinity to GDI. Therefore, the AXA box contributes to increase Rab binding affinity to GDI upon complex formation. | ||
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Finally, there is a highly conserved region in the CCR (residues 225-228) named the <scene name='Sandbox_208/Residues_225-228_ccr/2'>mobile effector loop (MEL)</scene>. This part of the CCR directs GDI to the membrane and regulated the ability of GDI to retrieve Rab to the cytosol. | Finally, there is a highly conserved region in the CCR (residues 225-228) named the <scene name='Sandbox_208/Residues_225-228_ccr/2'>mobile effector loop (MEL)</scene>. This part of the CCR directs GDI to the membrane and regulated the ability of GDI to retrieve Rab to the cytosol. | ||
*'''Domain II of GDI or Lipid Binding Site''': Domain II is rich in α helices. Helices D, E and H form a prenyl lipid binding pocket. <scene name='Sandbox_208/Lys145/2'>Lys145</scene> may play an important role in the lipid-binding cavity formation by functioning a spreader that keeps separated helices D and E. The GDI lipid binding pocket can adopt two different conformations, one being the open form when a lipid bound and the other being closed when neither lipid nor Rab is bound. A well ordered hydrophobic core stabilizes α helices D, E, F, G an H in a tighly packed state. This corresponds to the closed | *'''Domain II of GDI or Lipid Binding Site''': Domain II is rich in α helices. Helices D, E and H form a prenyl lipid binding pocket. <scene name='Sandbox_208/Lys145/2'>Lys145</scene> may play an important role in the lipid-binding cavity formation by functioning as a spreader that keeps separated helices D and E. The GDI lipid binding pocket can adopt two different conformations, one being the open form when a lipid bound and the other being closed when neither lipid nor Rab is bound. A well ordered hydrophobic core stabilizes α helices D, E, F, G an H in a tighly packed state. This corresponds to the closed conformation of the GDI lipid binding site. | ||
When GDI and Rab bind together, the vast majority of domain II retains its structure. However, there is a rearrangement in α helices, exposing a part of the hydrophobic core residues and forming a hydrophobic cleft on the GDI surface. Interactions between Rab and GDI via the CCR and RBP induce the lipid pocket opening. | When GDI and Rab bind together, the vast majority of domain II retains its structure. However, there is a rearrangement in α helices, exposing a part of the hydrophobic core residues and forming a hydrophobic cleft on the GDI surface. Interactions between Rab and GDI via the CCR and RBP induce the lipid pocket opening. | ||
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</StructureSection> | </StructureSection> | ||
= Catalytic mechanism = | = Catalytic mechanism<ref>PMID:16395334</ref> = | ||
[[Image:Catalytic mechanism RabGDI.jpg | thumb | 250 px | Model for the catalytic mechanism of RabGDI]] | [[Image:Catalytic mechanism RabGDI.jpg | thumb | 250 px | Model for the catalytic mechanism of RabGDI]] | ||
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The major effect of both mutations could lead to a decrease of the Rab pool available for synaptic vesicles cycling and neurotransmitter release. | The major effect of both mutations could lead to a decrease of the Rab pool available for synaptic vesicles cycling and neurotransmitter release. | ||
= External | = External resources = | ||
*[http://www.rcsb.org/pdb/explore.do?structureId=3CPH Crystal structure of Sec4 in complex with Rab-GDI] | |||
[http://www. | *[http://www.pdb.org/pdb/explore/explore.do?structureId=3CPJ Crystal structure of Ypt31 in complex with yeast Rab-GDI] | ||
=References= | |||
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= References = | |||
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