Fluoride Activation of the Rho Family GTP-binding Protein Cdc42Hs

Hoffman, Gregory R.; Nassar, Nicolas; Oswald, Robert E.; Cerione, Richard A.

doi:10.1074/jbc.273.8.4392

Cited by 43 publications

(49 citation statements)

References 35 publications

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“…These experiments were performed in the presence of excess GDP, and thermodynamic analysis of the binding isotherms shows that under these conditions, the apparent dissociation constant for Cdc42 binding to Cdc42GAP is on the order of 10 Ϯ 3 M and is the same for all 3 Cdc42 variants. This estimate for Cdc42 binding to Cdc42GAP in the presence of GDP agrees well with the dissociation constant of 2.8-2.9 M (24, 42) and is in reasonable agreement with the 50 M value reported by Hoffman et al (41). These experiments establish the feasibility of engineering a protein with enhanced stability without perturbing its enzymatic or signaling functions.…”

Section: Resultssupporting

confidence: 79%

Rational stabilization of enzymes by computational redesign of surface charge–charge interactions

Gribenko

Patel

Liu

et al. 2009

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

207

177

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Here, we report the application of a computational approach that allows the rational design of enzymes with enhanced thermostability while retaining full enzymatic activity. The approach is based on the optimization of the energy of charge-charge interactions on the protein surface. We experimentally tested the validity of the approach on 2 human enzymes, acylphosphatase (AcPh) and Cdc42 GTPase, that differ in size (98 vs. 198-aa residues, respectively) and tertiary structure. We show that the designed proteins are significantly more stable than the corresponding WT proteins. The increase in stability is not accompanied by significant changes in structure, oligomerization state, or, most importantly, activity of the designed AcPh or Cdc42. This success of the design methodology suggests that it can be universally applied to other enzymes, on its own or in combination with the other strategies based on redesign of the interactions in the protein core.Until man duplicates a blade of grass, nature will laugh at his so-called scientific knowledge.Thomas Edison computational design ͉ protein engineering ͉ protein stability R ational engineering of proteins to enhance stability and yet retain their enzymatic activity is well motivated (1). One motivation is the practical significance of expanding the use of enzymes in many areas of the modern world, including protein therapeutics, enzymes for food industry, diagnostics, and other areas of industrial biotechnology. Another motivation is validation of the existing scientific knowledge. In this case, predictions made by the existing models for protein stability are subjected to thorough experiments, testing their applicability to protein design. In this paper, we present the results of rational design of enzymes with enhanced stability and unchanged enzymatic activity. This approach has 2 major differences from previously described successful protein design methods (2-5): (i) it concentrates only on the residues on the protein surface, and (ii) it optimizes just one type of interactions, namely, charge-charge interactions on the protein surface (6-15).One of the most important aspects of engineering proteins with enhanced stability, retaining the enzymatic activity, is often forgotten. However, for all of these design efforts to be practically useful, it is important that the engineered proteins retain their biological and enzymatic activity. This issue is particularly important when enhanced protein stability is achieved by redesigning the charge-charge interactions on the protein surface. Such redesign can lead to several potentially detrimental effects on the activity: (i) it can affect the electrostatic potential in the active center, thus reducing or even abolishing the activity; (ii) it can affect substrate/product binding and again reduce or abolish the enzymatic activity; or (iii) it can have effects on the kinetics of substrate binding and, thus, lower the activity via reduced rates of electrostatic steering (3,(16)(17)(18)(19)(20)(21)(22).To this end, it is imp...

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Section: Resultssupporting

confidence: 79%

Rational stabilization of enzymes by computational redesign of surface charge–charge interactions

Gribenko

Patel

Liu

et al. 2009

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

207

177

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“…Purified GAP proteins were eluted as a single peak during a 0 -300 mM NaCl gradient in the same buffer, concentrated, and stored at Ϫ20°C in the presence of 40% glycerol. All fluorescence measurements were done on a T-format SLM 8000C Spectrofluorimeter, as described before (25)(26)(27) in 20 mM HEPES (pH 7.5), 5 mM MgCl 2 , and 100 mM NaCl.…”

Section: Methodsmentioning

confidence: 99%

“…Measurements of binding of the different GAPs to Cdc42Hs were done using the N-methylanthraniloyl (Mant) nucleotide assays described earlier (25,27). GTPase reaction rates were measured by monitoring the nucleotide-sensitive fluorescence from Trp 97 of Cdc42Hs (26).…”

Section: Methodsmentioning

confidence: 99%

“…Two basic spectroscopic approaches involving Mant-guanine nucleotides have been used; one involves environmental sensitivity, where protein-protein interactions that perturb the microenvironment of the Mant-moiety were shown to influence the fluorescence intensity of Mant-nucleotides bound to Ras, Cdc42Hs and Rac (25, 38 -41). A second approach involves taking advantage of the correlation between the polarized emission of a fluorophore and molecular size, to directly monitor complex formation between a fluorescently labeled protein and its binding partner (27,42). We have exchanged the nonhydrolyzable GTP analog, Mant-GppNHp into the active site of Cdc42Hs, and monitored Mant fluorescence intensity as a function of added wild-type or mutant Cdc42-GAP proteins.…”

Section: Cdc42hs (Y32k) Ndmentioning

confidence: 99%

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Biochemical Studies of the Mechanism of Action of the Cdc42-GTPase-activating Protein

Leonard

Lin

Cerione

et al. 1998

Journal of Biological Chemistry

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The small GTP-binding proteins Rac, Rho, and Cdc42 were shown to mediate a variety of signaling pathways including cytoskeletal rearrangements, cell-cycle progression, and transformation. Key to the proper function of these GTP-binding proteins is an efficient shutoff mechanism that ensures the decay of the signal. Regulatory proteins termed GAPs (GTPase-activating proteins) enhance the intrinsic GTP hydrolysis of the GTP-binding proteins, thereby ensuring signal termination. We have used site-specific mutagenesis to elucidate the limit domain for GAP activity in Cdc42-GAP, and show that in addition to the known GAP-homology domain (three conserved boxes), a C-terminal region outside that domain is also essential for GAP activity. In addition, we have replaced the conserved arginine (Arg 305 ), which was suggested by structural studies to be a key catalytic residue, with an alanine and found that the R305A Cdc42-GAP mutant has a greatly diminished catalytic capacity but is still able to bind Cdc42 with high affinity. Thus, a key catalytic role for this residue is confirmed. However, we also present evidence for the involvement of an additional residue(s), since the R305A Cdc42-GAP mutant still exhibits measurable activity. Some of this residual activity might result from a neighboring arginine, since a double mutant R305A/R306A shows a further decrease in catalytic activity.

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“…A number of tools have been used to study these proteins, including aluminum fluoride (AlF). AlF can enter live cells and bind to GDP-bound proteins, producing a complex structure with the nucleotide resembling the GTP-bound form of the molecule (or the GTP hydrolysis transition state) (11)(12)(13). AlF can induce relocalization of some GTPases to their target membranes (14,15).…”

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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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Fluoride Activation of the Rho Family GTP-binding Protein Cdc42Hs

Cited by 43 publications

References 35 publications

Rational stabilization of enzymes by computational redesign of surface charge–charge interactions

Rational stabilization of enzymes by computational redesign of surface charge–charge interactions

Biochemical Studies of the Mechanism of Action of the Cdc42-GTPase-activating Protein

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

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