GTPase mechanism and function: new insights from systematic mutational analysis of the phosphate-binding loop residue Ala30 of Rab5

Liang, Zong-Qi; Mather, Timothy; Li, Guangpu

doi:10.1042/bj3460501

Cited by 23 publications

(24 citation statements)

References 44 publications

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“…In the Rab5a GTPase, mutation of Ala30, which occupies the place of EF-Tu D21, to proline abolished the GTPase activity, suggesting an important catalytic role for this P-loop backbone amide group (49). Therefore, one further experiment that could have strengthened our conclusions would be to test the importance of the hydrogen bond between the backbone nitrogen atom of D21 and the β-γ bridging oxygen of GTP by mutating D21 to Pro.…”

Section: Resultsmentioning

confidence: 99%

Ribosome-induced tuning of GTP hydrolysis by a translational GTPase

Maracci

Peske

Dannies

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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GTP hydrolysis by elongation factor Tu (EF-Tu), a translational GTPase that delivers aminoacyl-tRNAs to the ribosome, plays a crucial role in decoding and translational fidelity. The basic reaction mechanism and the way the ribosome contributes to catalysis are a matter of debate. Here we use mutational analysis in combination with measurements of rate/pH profiles, kinetic solvent isotope effects, and ion dependence of GTP hydrolysis by EF-Tu off and on the ribosome to dissect the reaction mechanism. Our data suggest that-contrary to current models-the reaction in free EF-Tu follows a pathway that does not involve the critical residue H84 in the switch II region. Binding to the ribosome without a cognate codon in the A site has little effect on the GTPase mechanism. In contrast, upon cognate codon recognition, the ribosome induces a rearrangement of EF-Tu that renders GTP hydrolysis sensitive to mutations of Asp21 and His84 and insensitive to K + ions. We suggest that Asp21 and His84 provide a network of interactions that stabilize the positions of the γ-phosphate and the nucleophilic water, respectively, and thus play an indirect catalytic role in the GTPase mechanism on the ribosome.

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Section: Resultsmentioning

confidence: 99%

Ribosome-induced tuning of GTP hydrolysis by a translational GTPase

Maracci

Peske

Dannies

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…hGBP-1 (5 g) was incubated in 10 l of reaction buffer containing 10% glycerol, 0.1 mM DTT, and 6.6 mM (0.2 Ci) [␣-32 P]GTP (PerkinElmer) for 2 h at 37°C. SDS (2.5%) containing 10 mM EDTA was added to stop the reaction, and GTP, GDP, and GMP were separated by thin-layer chromatography on polyethyleneimine-cellulose sheets (J. T. Baker), as described (30). GTP binding was measured by using GTP-agarose beads (Sigma-Aldrich) equilibrated in GTPase reaction buffer: 25-l beads were rotated at 4°C with 5 g of hGBP-1 in a total volume of 100 l for 30 min.…”

Section: Methodsmentioning

confidence: 99%

Golgi targeting of human guanylate-binding protein-1 requires nucleotide binding, isoprenylation, and an IFN-γ-inducible cofactor

Modiano

Cresswell

2005

Proc. Natl. Acad. Sci. U.S.A.

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Human guanylate-binding protein-1 (hGBP-1) is a large GTPase, similar in structure to the dynamins. Like many smaller GTPases of the Ras͞Rab family, it is farnesylated, suggesting it may dock into membranes and perhaps play a role in intracellular trafficking. To date, however, hGBP-1 has never been associated with a specific intracellular compartment. Here we present evidence that hGBP-1 can associate with the Golgi apparatus. Redistribution from the cytosol to the Golgi was observed by immunofluorescence and subcellular fractionation after aluminum fluoride treatment, suggesting that it occurs when hGBP-1 is in its GTP-bound state. Relocalization was blocked by a farnesyl transferase inhibitor. The C589S mutant of hGBP-1, which cannot be farnesylated, and the previously uncharacterized R48P mutant, which cannot bind GTP, both failed to localize to the Golgi. These two mutants had a dominant-negative effect, preventing endogenous wild-type hGBP-1 from efficiently redistributing after aluminum fluoride treatment. Furthermore, hGBP-1 requires another IFN-␥-induced factor to be targeted to the Golgi, because constitutively expressed hGBP-1 remained cytosolic in cells treated with aluminum fluoride unless the cells were preincubated with IFN-␥. Finally, two nonhydrolyzing mutants of hGBP-1, corresponding to active mutants of Ras family proteins, failed to constitutively associate with the Golgi; we propose three possible explanations for this surprising result.antiviral proteins ͉ GTPases ͉ innate immunity

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“…The resulting variants have been analyzed for GTP hydrolysis, GTP binding, GTP dissociation, and biological activity. At position 30 [13], only the substitution with proline reduces the GTPase activity significantly (at least 12-fold) (35). Whereas most of the other substitutions at this position show either a small negative effect or no effect on the GTPase activity, the arginine substitution stimulates the intrinsic GTP hydrolysis by 5-fold.…”

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confidence: 99%

“…Whereas most of the other substitutions at this position show either a small negative effect or no effect on the GTPase activity, the arginine substitution stimulates the intrinsic GTP hydrolysis by 5-fold. It was proposed that this introduced arginine residue may mimic the function of an arginine finger motif (35), which enhances GTPase activity in trimeric GTPases (36,37) and small GTPase-GAP complexes (38) by positioning the positively charged guanidinium group close to the GTP ␥-phosphate group.…”

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confidence: 99%

“…Mutations at Ras Gly [12] often reduce its GTPase activity and increase its biological activity in cellular transformation (33); however, some of these variations can be found at the equivalent position in other functional WT small GTPases. To investigate structural roles of P-loop in GTP hydrolysis and biological function, Rab5a has been used as a model system to replace both Ser 29 [12] and Ala 30 [13] with all the other 19 amino acids (34,35). The choice of mutation sites was partly made based on their proximity to the ␥-phosphate group of the substrate GTP.…”

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confidence: 99%

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

Zhu¹,

Liu²,

Terzyan³

et al. 2003

Journal of Biological Chemistry

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GTPase domain crystal structures of Rab5a wild type and five variants with mutations in the phosphate-binding loop are reported here at resolutions up to 1.5 Å. Of particular interest, the A30P mutant was crystallized in complexes with GDP, GDP؉AlF 3 , and authentic GTP, respectively. The other variant crystals were obtained in complexes with a non-hydrolyzable GTP analog, GppNHp. All structures were solved in the same crystal form, providing an unusual opportunity to compare structures of small GTPases with different catalytic rates. The A30P mutant exhibits dramatically reduced GTPase activity and forms a GTP-bound complex stable enough for crystallographic analysis. Importantly, the A30P structure with bound GDP plus AlF 3 has been solved in the absence of a GTPase-activating protein, and it may resemble that of a transition state intermediate. Conformational changes are observed between the GTP-bound form and the transition state intermediate, mainly in the switch II region containing the catalytic Gln 79 residue and independent of A30P mutationinduced local alterations in the P-loop. The structures suggest an important catalytic role for a P-loop backbone amide group, which is eliminated in the A30P mutant, and support the notion that the transition state of GTPase-mediated GTP hydrolysis is of considerable dissociative character.As essential regulators of intracellular vesicle trafficking between subcellular compartments of eukaryotic cells, Rab proteins comprise the largest branch in the monomeric Ras-related GTPase superfamily (1, 2) and mediate membrane fusion and possibly vesicle budding as well (3-7). This group of 20 -25 kDa proteins share ϳ30% amino acid sequence identity (8). Like other Ras-related GTPases (small GTPases), Rab proteins serve as molecular switches by cycling between GTP-bound (on/active) and GDP-bound (off/inactive) conformations. Upon GTP binding, an extensive hydrophobic interface forms between two so-called switch regions (I and II) (9), resulting in presentation of ordered structural features characteristic for the active state that binds and responds to effectors/regulators (10, 11). The inactive form usually has displaced and mobile switch regions (11,12). The off-to-on process requires dissociation of GDP, which is an intrinsically slow and reversible process, and association of GTP. This process can be accelerated by guanidine nucleotide exchange factors (GEF) (13,14) and regulated by other proteins such as GDP dissociation inhibitors (GDI) (15). The on-to-off process is also an intrinsically slow but irreversible process, which involves hydrolysis of GTP to GDP and is stimulated by GTPase-activating proteins (GAP) 1 (16 -20). Despite the conserved catalytic machinery, the intrinsic GTP hydrolytic rates in the Rab family vary by more than an order of magnitude. For example, Rab5a exhibits a rate 20-fold higher than that of Rab6 or Rab7 (21). The intrinsic GTP hydrolytic rate of a GTPase is important for the association duration with its GTP-specific partners, and thus for it...

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GTPase mechanism and function: new insights from systematic mutational analysis of the phosphate-binding loop residue Ala30 of Rab5

Cited by 23 publications

References 44 publications

Ribosome-induced tuning of GTP hydrolysis by a translational GTPase

Ribosome-induced tuning of GTP hydrolysis by a translational GTPase

Golgi targeting of human guanylate-binding protein-1 requires nucleotide binding, isoprenylation, and an IFN-γ-inducible cofactor

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

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