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Mode of interaction of the Gαo subunit of heterotrimeric G proteins with the GoLoco1 motif of Drosophila Pins is determined by guanine nucleotides.

Lüchtenborg AM, Purvanov V, Melnik BS, Becker S, Katanaev VL - Biosci. Rep. (2015)

Bottom Line: Our data suggest that the orientation of the GoLoco1 motif on Gαo significantly differs between the two nucleotide states of the latter.In other words, a rotation of the GoLoco1 peptide in respect with Gαo must accompany the nucleotide exchange in Gαo.Our data have important implications for the mechanisms of Pins regulation in the process of asymmetric cell divisions.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Lausanne, CH-1011, Switzerland.

No MeSH data available.


Related in: MedlinePlus

GoLoco1 binds with similar affinity but different orientation to Gαo-GTP and Gαo-GDP(A) Sequence of the GoLoco1 peptide of Pins and schematic representation of its dansylated variant, with the dansyl group attached to the side chain of Lys1 of this sequence. The DQR triad conserved among GoLoco domains and mediating the interaction with the guanine nucleotide within Gα is shown in italics. (B and C) Saturation curves of binding of increasing concentrations of dansyl-GoLoco1 to Gαo preloaded with GDP or GTPγS as measured by FRET of tryptophan to dansyl indicate comparable binding of Gαo-GDP and Gαo-GTPγS to the peptide. The data is presented as arbitrary fluorescence units (B) and normalized fluorescence units (C). (D) Competition experiment using increasing concentrations of the unlabelled GoLoco1 peptide decreasing the normalized FRET signal from Gαo-GDP and Gαo-GTPγS complexes with the dansylated peptide. All the data are presented as mean ± S.E.M., n=7.
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Figure 1: GoLoco1 binds with similar affinity but different orientation to Gαo-GTP and Gαo-GDP(A) Sequence of the GoLoco1 peptide of Pins and schematic representation of its dansylated variant, with the dansyl group attached to the side chain of Lys1 of this sequence. The DQR triad conserved among GoLoco domains and mediating the interaction with the guanine nucleotide within Gα is shown in italics. (B and C) Saturation curves of binding of increasing concentrations of dansyl-GoLoco1 to Gαo preloaded with GDP or GTPγS as measured by FRET of tryptophan to dansyl indicate comparable binding of Gαo-GDP and Gαo-GTPγS to the peptide. The data is presented as arbitrary fluorescence units (B) and normalized fluorescence units (C). (D) Competition experiment using increasing concentrations of the unlabelled GoLoco1 peptide decreasing the normalized FRET signal from Gαo-GDP and Gαo-GTPγS complexes with the dansylated peptide. All the data are presented as mean ± S.E.M., n=7.

Mentions: Previously we have demonstrated that the GoLoco1 domain of Drosophila Pins efficiently interacts with both the GDP-loaded and the GTP-loaded form of Gαo [5]. In order to investigate the unusual interaction of the GoLoco1 domain with Gαo-GTP in detail, we performed a series of FRET experiments. To this end, we synthetized the extended 35 amino acid long Pins GoLoco1 peptide and its fluorescent analogue, where the side chain of the N-terminal lysine was dansylated (dGoLoco1, Figure 1A). The dansyl group added on proteins/peptides has been extensively used as an acceptor of energy from excited tryptophans in FRET experiments [15,16]. We first confirmed the binding of GoLoco1 with Gαo, taking advantage of the fact that FRET occurs between the donor tryptophan and the acceptor dansyl in case donor and acceptor are in close proximity. Upon excitement of tryptophan, a FRET signal should therefore only been seen upon binding of dGoLoco1 to Gαo, and the strength of the signal should be proportional to the distance between the tryptophan donor and the dansyl acceptor. Indeed, we detect a robust FRET signal to dGoLoco1 from both nucleotide states of Gαo, although the signal from Gαo-GTPγS was several folds lower than from Gαo-GDP (Figure 1B). However, analysis of the saturation curves shows that the affinity of dGoLoco1 to the two nucleotide forms of Gαo is similar (Figure 1C): Kd=4.6 μM for Gαo-GDP–dGoLoco1 and Kd=2.1 μM for Gαo-GTPγS–dGoLoco1. Further, dGoLoco1 from both complexes can be similarly outcompeted by the non-dansylated GoLoco1 peptide (Figure 1D). The Ki values resulting from these competition experiments are 17.6 μM for Gαo-GDP and 10.6 μM for Gαo-GTPγS. The finding that Ki for unlabelled GoLoco1 is somewhat higher than Kd for dGoLoco1 may indicate that the hydrophobic dansyl group increases the affinity of the GoLoco1 peptide to Gαo. In any regard, we conclude that the binding affinity of the GoLoco1 peptide of Pins to Gαo is similar for the two nucleotide states of the G protein, but that the proximity of the N-terminus of GoLoco1 to Gαo differs in the two nucleotide states.


Mode of interaction of the Gαo subunit of heterotrimeric G proteins with the GoLoco1 motif of Drosophila Pins is determined by guanine nucleotides.

Lüchtenborg AM, Purvanov V, Melnik BS, Becker S, Katanaev VL - Biosci. Rep. (2015)

GoLoco1 binds with similar affinity but different orientation to Gαo-GTP and Gαo-GDP(A) Sequence of the GoLoco1 peptide of Pins and schematic representation of its dansylated variant, with the dansyl group attached to the side chain of Lys1 of this sequence. The DQR triad conserved among GoLoco domains and mediating the interaction with the guanine nucleotide within Gα is shown in italics. (B and C) Saturation curves of binding of increasing concentrations of dansyl-GoLoco1 to Gαo preloaded with GDP or GTPγS as measured by FRET of tryptophan to dansyl indicate comparable binding of Gαo-GDP and Gαo-GTPγS to the peptide. The data is presented as arbitrary fluorescence units (B) and normalized fluorescence units (C). (D) Competition experiment using increasing concentrations of the unlabelled GoLoco1 peptide decreasing the normalized FRET signal from Gαo-GDP and Gαo-GTPγS complexes with the dansylated peptide. All the data are presented as mean ± S.E.M., n=7.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4660580&req=5

Figure 1: GoLoco1 binds with similar affinity but different orientation to Gαo-GTP and Gαo-GDP(A) Sequence of the GoLoco1 peptide of Pins and schematic representation of its dansylated variant, with the dansyl group attached to the side chain of Lys1 of this sequence. The DQR triad conserved among GoLoco domains and mediating the interaction with the guanine nucleotide within Gα is shown in italics. (B and C) Saturation curves of binding of increasing concentrations of dansyl-GoLoco1 to Gαo preloaded with GDP or GTPγS as measured by FRET of tryptophan to dansyl indicate comparable binding of Gαo-GDP and Gαo-GTPγS to the peptide. The data is presented as arbitrary fluorescence units (B) and normalized fluorescence units (C). (D) Competition experiment using increasing concentrations of the unlabelled GoLoco1 peptide decreasing the normalized FRET signal from Gαo-GDP and Gαo-GTPγS complexes with the dansylated peptide. All the data are presented as mean ± S.E.M., n=7.
Mentions: Previously we have demonstrated that the GoLoco1 domain of Drosophila Pins efficiently interacts with both the GDP-loaded and the GTP-loaded form of Gαo [5]. In order to investigate the unusual interaction of the GoLoco1 domain with Gαo-GTP in detail, we performed a series of FRET experiments. To this end, we synthetized the extended 35 amino acid long Pins GoLoco1 peptide and its fluorescent analogue, where the side chain of the N-terminal lysine was dansylated (dGoLoco1, Figure 1A). The dansyl group added on proteins/peptides has been extensively used as an acceptor of energy from excited tryptophans in FRET experiments [15,16]. We first confirmed the binding of GoLoco1 with Gαo, taking advantage of the fact that FRET occurs between the donor tryptophan and the acceptor dansyl in case donor and acceptor are in close proximity. Upon excitement of tryptophan, a FRET signal should therefore only been seen upon binding of dGoLoco1 to Gαo, and the strength of the signal should be proportional to the distance between the tryptophan donor and the dansyl acceptor. Indeed, we detect a robust FRET signal to dGoLoco1 from both nucleotide states of Gαo, although the signal from Gαo-GTPγS was several folds lower than from Gαo-GDP (Figure 1B). However, analysis of the saturation curves shows that the affinity of dGoLoco1 to the two nucleotide forms of Gαo is similar (Figure 1C): Kd=4.6 μM for Gαo-GDP–dGoLoco1 and Kd=2.1 μM for Gαo-GTPγS–dGoLoco1. Further, dGoLoco1 from both complexes can be similarly outcompeted by the non-dansylated GoLoco1 peptide (Figure 1D). The Ki values resulting from these competition experiments are 17.6 μM for Gαo-GDP and 10.6 μM for Gαo-GTPγS. The finding that Ki for unlabelled GoLoco1 is somewhat higher than Kd for dGoLoco1 may indicate that the hydrophobic dansyl group increases the affinity of the GoLoco1 peptide to Gαo. In any regard, we conclude that the binding affinity of the GoLoco1 peptide of Pins to Gαo is similar for the two nucleotide states of the G protein, but that the proximity of the N-terminus of GoLoco1 to Gαo differs in the two nucleotide states.

Bottom Line: Our data suggest that the orientation of the GoLoco1 motif on Gαo significantly differs between the two nucleotide states of the latter.In other words, a rotation of the GoLoco1 peptide in respect with Gαo must accompany the nucleotide exchange in Gαo.Our data have important implications for the mechanisms of Pins regulation in the process of asymmetric cell divisions.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Lausanne, CH-1011, Switzerland.

No MeSH data available.


Related in: MedlinePlus