Sandbox 208: Difference between revisions
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In cells, vesicular traffic through the exocytic and endocytic pathways involves the activity of small Rab superfamilly [http://http://en.wikipedia.org/wiki/GTPase GTPases] which are named [http://http://en.wikipedia.org/wiki/Rab_(G-protein) Rab proteins]. Rab proteins exist in two different conformations: an active GTP bound conformation and an inactive GDP bound conformation. The process of switching between these two conformations requires a multitude of interacting and also regulatory proteins. Rab active form interacts with its effectors i.e GTPases activating proteins. On the contrary, the inactive conformation is recognized by guanine nucleotide exchange factors ([http://http://en.wikipedia.org/wiki/Guanine_nucleotide_exchange_factor GEF]) and regulators proteins including [http://http://en.wikipedia.org/wiki/Guanosine_nucleotide_dissociation_inhibitors GDI]. GDI is an inhibitory protein which extracts prenylated Rab proteins from membranes at the end of their cycle of activity and facilitates their delivery to the donor membranes. GDI is indispensable for the vesicular transport machinery functioning: its deletion is lethal. | In cells, vesicular traffic through the exocytic and endocytic pathways involves the activity of small Rab superfamilly [http://http://en.wikipedia.org/wiki/GTPase GTPases] which are named [http://http://en.wikipedia.org/wiki/Rab_(G-protein) Rab proteins]. Rab proteins exist in two different conformations: an active GTP bound conformation and an inactive GDP bound conformation. The process of switching between these two conformations requires a multitude of interacting and also regulatory proteins. Rab active form interacts with its effectors i.e GTPases activating proteins. On the contrary, the inactive conformation is recognized by guanine nucleotide exchange factors ([http://http://en.wikipedia.org/wiki/Guanine_nucleotide_exchange_factor GEF]) and regulators proteins including [http://http://en.wikipedia.org/wiki/Guanosine_nucleotide_dissociation_inhibitors GDI]. GDI is an inhibitory protein which extracts prenylated Rab proteins from membranes at the end of their cycle of activity and facilitates their delivery to the donor membranes. GDI is indispensable for the vesicular transport machinery functioning: its deletion is lethal. | ||
In this entry, we only focus on the structure of the complex between GDI and a doubly [http://http://en.wikipedia.org/wiki/Prenylated prenylated] Rab proteins. | In this entry, we only focus on the structure of the complex between GDI and a doubly [http://http://en.wikipedia.org/wiki/Prenylated prenylated] Rab proteins.<ref>PMID:18426803</ref> | ||
= Biological function = | = Biological function<ref>PMID:12623022</ref> <ref>PMID:15924270</ref> <ref>PMID: 12802060</ref> = | ||
*'''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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Both REP and GDI are considered as Rab molecular [http://http://en.wikipedia.org/wiki/Chaperone_(protein) chaperones]. They have similar organization, in particular, their Rab binding interface which is well conserved. | Both REP and GDI are considered as Rab molecular [http://http://en.wikipedia.org/wiki/Chaperone_(protein) chaperones]. They have similar organization, in particular, their Rab binding interface which is well conserved. | ||
=Structure= | =Structure<ref>PMID: 18426803</ref>= | ||
<StructureSection load='3cpi' size='550' side='right' caption='Assymetric unit of Rab-GDP Dissociation Inhibitor (PDB entry [[3cpi]])' scene=''> | <StructureSection load='3cpi' size='550' side='right' caption='Assymetric unit of Rab-GDP Dissociation Inhibitor (PDB entry [[3cpi]])' scene=''> | ||
3CPI is a 2 chains structure of sequences from [http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae Saccharomyces cerevisiae]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3CPI OCA]. | 3CPI is a 2 chains structure of sequences from [http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae Saccharomyces cerevisiae]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3CPI OCA]. | ||
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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/ | *'''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. | ||
Finally, there is a highly conserved region in the CCR (residues 225-228) named the <scene name='Sandbox_208/Residues_225-228_ccr/ | 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/ | *'''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. | ||
Both geranylgeranyl (GG) moities are located in the lipid binding site on top of each other. In this arrangement, the first bent lipid (GG1) protrudes into the core of domain II, anchoring to the lipid binding site thanks to the GDI residues <scene name='Sandbox_208/Gg1/ | Both geranylgeranyl (GG) moities are located in the lipid binding site on top of each other. In this arrangement, the first bent lipid (GG1) protrudes into the core of domain II, anchoring to the lipid binding site thanks to the GDI residues <scene name='Sandbox_208/Gg1/2'>Val127, Pro128, Ala129, Ala134, Leu139, Met140, Met148, Leu152, Phe192, Met197, Cys221, Val224 and Ala225</scene>. | ||
The second geranylgeranyl group (GG2) is located on the surface and it is aligned between helices D and E. Its binding site involves only seven hydrophobic amino acids: <scene name='Sandbox_208/Gg2/ | The second geranylgeranyl group (GG2) is located on the surface and it is aligned between helices D and E. Its binding site involves only seven hydrophobic amino acids: <scene name='Sandbox_208/Gg2/2'>Met148, Leu152, Ile155, Ile193, Trp200, Tyr205 and Leu218</scene>. GG2 forms a lid shielding a large part of the buried GG1 from the solvent. The environnement of GG2 is more hydrophilic than of the buried lipid. | ||
GDIs have functional specificity for a particular Rab. Both yeast and mammalians GDIs contains specific residues that contribute to the strength of their interaction with distinct Rab species. | GDIs have functional specificity for a particular Rab. Both yeast and mammalians GDIs contains specific residues that contribute to the strength of their interaction with distinct Rab species. | ||
</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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Finally, the Rab protein enters its functional cycle whereas GDI is released into the cytosol. | Finally, the Rab protein enters its functional cycle whereas GDI is released into the cytosol. | ||
= RabGDI deficiency and diseases = | = RabGDI deficiency and diseases<ref>PMID:21736009</ref> <ref>PMID: 12623022</ref> = | ||
GDI1, one of the genes involved in the control of cycling between active and inactive state of the Rab family, has a major role in mental disorder. In fact, Gdi1 encodes α-GDI, which is specific for Rab3. Rab3 plays an important role in neurotransmitter release. Therefore, mutations in GDI1 cause functional and developmental alterations in the neuron which could lead to a severe impairment of learning abilities. More precisely, mutations in GDI1 are linked to X-linked nonspecific mental retardation. Two kinds of alterations in the GDI1 gene could be found and they determine two families affected with X-linked non-specific mental retardation (MRX48 and MRX41). | GDI1, one of the genes involved in the control of cycling between active and inactive state of the Rab family, has a major role in mental disorder. In fact, Gdi1 encodes α-GDI, which is specific for Rab3. Rab3 plays an important role in neurotransmitter release. Therefore, mutations in GDI1 cause functional and developmental alterations in the neuron which could lead to a severe impairment of learning abilities. More precisely, mutations in GDI1 are linked to X-linked nonspecific mental retardation. Two kinds of alterations in the GDI1 gene could be found and they determine two families affected with X-linked non-specific mental retardation (MRX48 and MRX41). | ||
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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 resources = | ||
*[http://www.rcsb.org/pdb/explore.do?structureId=3CPH Crystal structure of Sec4 in complex with Rab-GDI] | |||
= References = | *[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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