High Resolution Crystal Structures of Human Rab5a and Five Mutants with Substitutions in the Catalytically Important Phosphate-binding Loop

Many GTPases regulate intracellular transport and signaling in eukaryotes. Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing the exchange of their GDP for GTP. Here we present crystallographic and biochemical studies of a GEF reaction with four crystal structures of Arabidopsis thaliana ARA7, a plant homolog of Rab5 GTPase, in complex with its GEF, VPS9a, in the nucleotide-free and GDP-bound forms, as well as a complex with aminophosphonic acid-guanylate ester and ARA7⅐VPS9a(D185N) with GDP. Upon complex formation with ARA7, VPS9 wedges into the interswitch region of ARA7, inhibiting the coordination of Mg 2؉ and decreasing the stability of GDP binding. The aspartate finger of VPS9a recognizes GDP ␤-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP ␤-phosphate toward switch II to further destabilize GDP for its release during the transition from the GDP-bound to nucleotide-free intermediates in the nucleotide exchange reaction.Small GTPases work as a molecular switch, which is turned off by its intrinsic GTPase activity hydrolyzing GTP to GDP. To turn the switch on, the bound GDP should be removed to introduce a GTP. Normally, this exchange reaction is much slower than the rate of intrinsic GTPase activity because nucleotide binds to small GTPase tightly with the Mg 2ϩ ion. GEF 5 enhances the nucleotide exchange of its cognate GTPase by destabilizing the Mg 2ϩ ion and GDP binding to the GTPase. Recognition of the GDP-bound GTPase by GEF is a critical but transient step prior to the formation of a nucleotide-free GTPase⅐GEF binary complex (1, 2). Because the nucleotide-free complex is stable in vitro, most GTPase⅐GEF complexes have been crystallized and structurally analyzed as the nucleotidefree form in the past, with a few exceptions containing nucleotides. One of them is a structure of eFF1A⅐eEF1B␣ in complex with different di-and triphosphate nucleotides, in which ␤-and ␥-phosphate electron densities of the nucleotides were ambiguous and only GMP could have been modeled (3). Another example is the Arf1⅐Sec7 complex, in which introducing either an abortive inhibitor or a mutation on the GEF allowed for GDP binding into the Arf1⅐Sec7 complex (4, 5). The complex structure between ROP4⅐GDP, a member of the plant Rho family, and its GEF PRONE8 indicated that GDP bound to the ROP4⅐PRONE8 complex loosely (6). A complex between Cdc42 and the DHR2 GEF domain of DOCK9 GEF has been crystallized in three different nucleotide forms: nucleotide-free, GDPbound, and GTP/Mg 2ϩ -bound. These structures demonstrate that the nucleotide sensor found in the ␣10 helix of the DHR2 domain is responsible for the exclusion of GDP/Mg 2ϩ and the introduction of GTP/Mg 2ϩ (7). It should be noted that none of the nucleotides were directly recognized by the GEFs in the complex structures mentioned above. These exceptions have encouraged us to investigate the transient nucleotide recognition in the GEF-catalyzed reaction.Rab small GTPases regulate vesicular transport in eukaryotes (8, 9), including ...

Section: Resultsmentioning

confidence: 81%

“…4a). Interestingly, a side-chain oxygen of the glutamate finger, Gea2 Glu 654 , also forms a salt bridge with Arf1 Lys 30 , corresponding to ARA7 Lys 23 .…”

Section: Resultsmentioning

confidence: 99%

GDP-bound and Nucleotide-free Intermediates of the Guanine Nucleotide Exchange in the Rab5·Vps9 System

Uejima

Ihara

Goh

et al. 2010

“…However, these mutants remain prenylated, membrane-associated, and functionally interact with guanine nucleotides (40,41,44). In addition, functional aspects of protein folding and the crystal structure also remain unaffected (44,45). Therefore, the T7A mutation would not be predicted to interfere with the GTPase activity of Rab5a and should not affect its three-dimensional conformation.…”

Section: T7a Mutation Partially Inhibits Rab5amentioning

confidence: 99%

Phosphorylation of Rab5a Protein by Protein Kinase Cϵ Is Crucial for T-cell Migration

Ong

Freeley

Skubis-Zegadło

et al. 2014

Background: Rab5a GTPase plays important roles in intracellular transport and cell signaling. Results: T-cell stimulation through the integrin LFA-1 or the chemokine receptor CXCR4 induces PKC⑀-dependent phosphorylation of Rab5a at Thr-7, which is crucial for cytoskeleton remodeling and cell migration. Conclusion: PKC⑀-Rab5a-Rac1 axis regulates T-cell motility. Significance: The study provides novel insights into the role of Rab5a in the adaptive immune response.

“…Recent structural studies of the GDP/GTP cycle of Rab proteins have shown that, despite their non-conventional cellular cycle, they do not depart from other small GTP-binding proteins and respond to the alternation of GDP and GTP by conformational changes and disorder-to-order transitions at the so-called switch 1 and switch 2 regions (28,29). These studies of their GDP/GTP cycles, together with the structure of the complex of Rab3 with its effector Rabphilin3 (30), the structures of Rab proteins bound to either GDP (31) or GTP analogues (32)(33)(34)(35), and genome-wide analysis of Rab sequences (5,6), suggest that the structural specificity of Rab subfamilies resides in the combination of the nucleotide-sensitive switch regions with nucleotide-insensitive regions featuring subfamily specific sequences and/or conformations. These latter regions have been termed RabSF1-4 and include the N terminus/␤1, ␣1/switch 1, ␣3-␤5, and ␣5/C terminus, respectively (6).…”

mentioning

confidence: 99%

The Structural GDP/GTP Cycle of Rab11 Reveals a Novel Interface Involved in the Dynamics of Recycling Endosomes

Pasqualato

Senic-Matuglia²,

Renault³

et al. 2004

105

The small GTP-binding protein Rab11 is an essential regulator of the dynamics of recycling endosomes. Here we report the crystallographic analysis of the GDP/GTP cycle of human Rab11a, and a structure-based mutagenesis study that identifies a novel mutant phenotype. The crystal structures show that the nucleotide-sensitive switch 1 and 2 regions differ from those of other Rab proteins. In Rab11-GDP, they contribute to a close packed symmetrical dimer, which may associate to membranes in the cell and allow Rab11 to undergo GDP/ GTP cycles without recycling to the cytosol. The structure of active Rab11 delineates a three-dimensional site that includes switch 1 and is separate from the site defined by the Rab3/Rabphilin interface. It is proposed to form a novel interface for a Rab11 partner compatible with the simultaneous binding of another partner at the Rabphilin interface. Mutation of Ser 29 to Phe in this epitope resulted in morphological modifications of the recycling compartment that are distinct from those induced by the classical dominant-negative and constitutively active Rab11 mutants. Recycling endosomes condensed in the perinuclear region where they retained recycling transferrin, and they clustered Rab11-and EEA1-positive membranes. Altogether, our study suggests that this mutation impairs a specific subset of Rab11 interactions, possibly those involved in cytoskeleton-based movements driving the slow recycling pathway.