Sandbox 208

Crystal structure of yeast Rab-GDI

spinqualitylabelsall models PDB ID 3cpi Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image
3cpi, resolution 2.30Å ()
Related:	3cph, 3cpj
Structural annotation: Resources: UniProt : P39958
Evolutionary conservation: Toggle Conservation Colors: Rows = identical sequences: Further Information: Caveats, Explanation, Complete Results at ConSurfDB
Resources:	FirstGlance, OCA, RCSB, PDBsum
Coordinates:	save as pdb, mmCIF, xml

Rab-Guanosine biphosphate Dissociation Inhibitor (RabGDI) belongs to the transport cytoplasmic protein group. RabGDI was firstly isolated from bovine brain and three isoforms were isolated (α, β, γ). However, in the yeast Saccharomyces cerevisiae, only one isoform was identified: Gdi1p.

In cells, vesicular traffic through the exocytic and endocytic pathways involves the activity of small Rab superfamilly GTPases which are named 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 (GEF) and regulators proteins including 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 prenylated Rab proteins.

Biological function Biological function[2] [3] [4]

• An essential step: Rab prenylation

All Rabs undergo 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. Geranylgeranylation is catalyzed by the prenyltransferase, geranylgeranyltransferase type II (GGTrII), a heterodimer containing α- and β-subunits. GGTrII requires the activity of an accessory protein, Rab escort protein (REP). REP is able to bind newly synthesized Rabs and to mediate their delivery to GGTrII.

• GDI versus REP

GDI does not facilitate Rabs prenylation since it lacks the geranylgeranyl transferase binding site. It is involved in Rabs recycling. Its function consists in retrieving Rab in the GDP bound form from the membrane and delivers it to the cytosol. Thus, GDI controls the Rabs distribution between cytosol and membranes. GDI is stably associated only with GDP loaded and prenylated Rab proteins. Both REP and GDI are considered as Rab molecular chaperones. They have similar organization, in particular, their Rab binding interface which is well conserved.

StructureStructure[5]

3CPI is a 2 chains structure of sequences from Saccharomyces cerevisiae. Full crystallographic information is available from OCA. Overall structure GDI is a 55-kDa protein containing 451 residues. The structure of GDI contains 27 β strands and 21 α helices and can be divided into two domains separated by a cleft. Firstly, a large domain I is located at the apex. Secondly, a smaller entirely α helices domain named domain II is inserted at the base of GDI. The β sheets build a relatively rigid protein carcass. The α helical regions are involved in the structural rearrangement upon Rab 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 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-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 from domain I and 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 . 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. 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. 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 . 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: . 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.

spinqualitylabelsall models Assymetric unit of Rab-GDP Dissociation Inhibitor (PDB entry 3cpi) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Catalytic mechanismCatalytic mechanism[6]

Model for the catalytic mechanism of RabGDI

Initially, Rab is anchored to a target membrane via its lipid moieties. After interactions with its effectors, it returns to the GDP bound form. There is a primary recognition between Rab and GDI: the GTPase domain of Rab is recognized by GDI. GDI binds to Rab via the RBP, forming a low affinity complex. Then, the Rab conserved C terminal AXA box is recognized and bound by the GDI CCR, increasing the complex affinity. Interactions between CCR and the hydrophobic residues of the AXA box lead to a conformation change of the domain II. The GDI lipid binding pocket opens. It is located in the vicinity of the Rab lipid anchor, leading to a favorable situation for lipid transfer. Firstly, the first extracted lipid initially binds to the superficial lipid binding site leading to the formation of a transient high affinity complex still anchored in the membrane. Secondly, this complex is converted into a soluble complex by coordinating transfer of the GDI-bound lipid into the buried binding site. This event facilitates flipping of the second lipid from the membrane to the surface binding site. Thus, a high affinity Rab-GDI complex is formed and released from the membrane. GDI transports and then mediates the delivery of prenylated Rab to another target membrane. GDI leads to the docking of Rab via a protein interaction with the protein GDI. The docked complex undergoes a conformational change. This leads to the transfer of the first and then the second geranylgeranyl moiety into the membrane and subsequently to the release of the Rab C-terminus from the CBR. Finally, the Rab protein enters its functional cycle whereas GDI is released into the cytosol.

RabGDI deficiency and diseases RabGDI deficiency and diseases[7] [8]

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). In the family MRX48, there is a C -> T transition at position 366. The mutation disrupts synthesis of alpha-GDI by introducing a premature stop codon in the open reading frame. The mutation has a dominant phenotypic effect as carrier females in the family are also affected. The second mutation, in the MRX41 family, is a T -> C transition at position 433, causing a missense mutation and a non-conservative amino acid change (L92P). This mutation leads to a 6,3-fold decrease in affinity for Rab3A. The mutant α-GDI may not be able to efficiently recycle Rab proteins in vivo. The introduction of the helix-breaking proline residue at position 92 may either indirectly destabilize the adjacent Rab binding region or reduce the ability of alpha-GDI to recognize the C-terminus prenyl group required for high affinity binding during recycling. The major effect of both mutations could lead to a decrease of the Rab pool available for synaptic vesicles cycling and neurotransmitter release.

External resourcesExternal resources

• Crystal structure of Sec4 in complex with Rab-GDI

• Crystal structure of Ypt31 in complex with yeast Rab-GDI