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We know that GDI deletion can be lethal in yeast (*), whereas a mutation in the alpha-RabGDI gene, residue I100, leads to X-linked nonsyndromic mental retardation in humans. This mutation is characterized by reduced extraction from the membrane. This residue is located in the CCR and is part of a group of nonpolar residues on the surface of the RabGDI molecule that form an extended hydrophic patch with a central cavity on the lower part of domain II. This assembly is involved in binding of the C-terminus of Rab via interaction with Val191 and Leu193 and induces a 90° turn in the C-terminus, which directs it over the effector loop toward the lipid-binding site. Mutation is this hydrophobic patch are expected have a two fold effect. | We know that GDI deletion can be lethal in yeast (*), whereas a mutation in the alpha-RabGDI gene, residue I100, leads to X-linked nonsyndromic mental retardation in humans. This mutation is characterized by reduced extraction from the membrane. This residue is located in the CCR and is part of a group of nonpolar residues on the surface of the RabGDI molecule that form an extended hydrophic patch with a central cavity on the lower part of domain II. This assembly is involved in binding of the C-terminus of Rab via interaction with Val191 and Leu193 and induces a 90° turn in the C-terminus, which directs it over the effector loop toward the lipid-binding site. Mutation is this hydrophobic patch are expected have a two fold effect. | ||
First, they will | First, they will impair C-terminus binding and will reduce the affinity of the RabGDI molecule for Rab. Second, and probably more important, they will perturb the orientation of the Rab C-terminus in the vicinity of the effector loop and the lipid-binding domain. This is likely interfere with GTPase interaction with molecules assisting delivery and removal of Rab proteins to and from the membrane. | ||
= Additional 3D Structures of Rab GDP-Dissociation Inhibitor = | = Additional 3D Structures of Rab GDP-Dissociation Inhibitor = | ||
Revision as of 16:08, 3 December 2011
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Rab-Guanosine biphosphate Dissociation Inhibitor (or Rab-GDI) is an inhibitory protein which facilitated extraction of prenylated GDP-bounds inactive conformation of Rab small GTPase from membranes. This molecule has an important role in vesicular membrane trafficking. It delivers Rab to new formes vesicles (for exocytic and endocytic pathways), where it becomes activated to the GTP-bound form to promote the recuitment of effectors that facilitate vesicle transport through the cytoplasm by the cytoskeleton. This inhibition can be removed by the action of a GEF.
Biological roleBiological role
Rab-GDI does not facilitate Rab prenylation, but serves as a generic regulator for recycling of Rab-GTPases for use in multiple rounds of membrane transport. It retrieves Rabs in the GDP-bound form from the membrane and delivers it to the cytosol, controlling the distribution of Rabs between membranes and cytosol. GDI is believed to be stably only with GDP-loaded and prenylated Rabs proteins, ensuring retrieval of inactivated Rab GTPases from the membrane at the end of their functionnal cycle. Rab-GDI is critically important for the proper functionning of the vesicular transport machinery, and its deletion can be lethal.
StructureStructure
General structureGeneral structure
Substrate bindingSubstrate binding
Contact between the molecules is etablished through a combination of polar et hydrophobic interactions and involves the switch I and II regions and the C-terminus of Rab, including the geranylgeranyl moiety.
GDI binds the Rab molecule via three interaction sites:
GDI-Rab Binding Platform (RBP), with bêta strands e1 and e3 and helix C, which form a separate binding site. It appears to be essential structural element, forming a 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), located in the cleft between domain I and domain II, which coordinates the flexible extended C-terminus of Rab. It is formed by residues 93-112 from domain I and 226-235 from domain II and reprent a hydrophobic cavity on the surface of the protein located between the GDI domains. Hydrophobic contacts between GDI and Rab are supported by a hydrogen bond involving main chain atoms.
Domain II of GDI or Lipid Binding Site, consisting solely of alpha helices D, E, H and F of domain II, which form a prenyl-lipid binding pocket, exhibiting an open conformation and accomodating the prenyl moiety of a modified Rab if present. K145 (on GDI) may play an important role in formation of lipid-binding cavity by functionning as a spreader that keeps helices D and E appart.
Additional minor contacts involve the N-terminus and C-terminus of GDI, as well as the Mobile Effector Loop (MEL). C-terminus of Rab molecules must be located in the vicinity of the MEL that is necessary for interaction with target membranes.
More localized interactions may also contribute to the affinity increase for GDP. Several residues of RabGDI establish contacts with residues of the switch I and II regions in the vicinity of the phosphate groups of GDP, in particular R248 (on GDI) forms a hydrogen bonds with the main chain oxygen od D63 (on Rab). This hydrogen bonds stabilizes the coordination of Mg2+ via water molecules, important for both nucleotide bonding and hydrolysis. It reduces the rate of GDP release.
Catalytic mechanismCatalytic mechanism
In the GDI domain I, the four helices A, C I and N forme a bundle. Helix I and the loop adjacent to the helix C belong to RBP and make direct contact with the globular core domain of the Rab molecule upon its binding. This relatively low affinity binding is followed by interaction of the initially disordered C-terminus with the hydrophobic patch of the CCR. Rab binding may promote the rearrangement of the GDI helices by pushing helix I toward the GDI core, whereas the loop following helix C is pushed away from the core, resulting in displacement of helices C and N. The displaced helices C and N make direct contact with domain II of GDI and appear to induce a conformationnal change, resulting in structural reorganization of domain II. The majority of GDI domain II (helices E, H, G and F) retains its structure upon Rab binding. This stabilizes the interaction of domain II of GDI with the membrane over the buried genranylgaranyl moities. However, there is a change in its orientation relative to domain I, and helix D is not tighly packed within domain II anymore. The side chain of Phe192 located in helix G flips and pushes the loop following helix D away, stabilizing the pocket in the open conformation. A conformational change leads to opening of the hydrophobic cavity between helices D and E in domain II and facilitate extraction of the geranylgeranyl lipid from the bilayer. Solvent exposure presumably is the reason for the moderate affinity of GDI to unprenylated Rab: the increase affinity of GDI to Rab upon prenyl group binding might be due to the Rab lipid moiety filling the open hydrophobic lipid binding pocket in GDI, diminishing the solvent exposed hydrophobic surfaces of the GDI-Rab complex. The large increase in affinity of GDI to Rab upon prenyl group binding appears to be the driving force for the membrane extraction process.
DiseasesDiseases
We know that GDI deletion can be lethal in yeast (*), whereas a mutation in the alpha-RabGDI gene, residue I100, leads to X-linked nonsyndromic mental retardation in humans. This mutation is characterized by reduced extraction from the membrane. This residue is located in the CCR and is part of a group of nonpolar residues on the surface of the RabGDI molecule that form an extended hydrophic patch with a central cavity on the lower part of domain II. This assembly is involved in binding of the C-terminus of Rab via interaction with Val191 and Leu193 and induces a 90° turn in the C-terminus, which directs it over the effector loop toward the lipid-binding site. Mutation is this hydrophobic patch are expected have a two fold effect. First, they will impair C-terminus binding and will reduce the affinity of the RabGDI molecule for Rab. Second, and probably more important, they will perturb the orientation of the Rab C-terminus in the vicinity of the effector loop and the lipid-binding domain. This is likely interfere with GTPase interaction with molecules assisting delivery and removal of Rab proteins to and from the membrane.