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Identification of Interactions between Abscisic Acid and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase.

Galka MM, Rajagopalan N, Buhrow LM, Nelson KM, Switala J, Cutler AJ, Palmer DR, Loewen PC, Abrams SR, Loewen MC - PLoS ONE (2015)

Bottom Line: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was identified as a putative ABA-binding protein.Overall we conclude that (+)-ABA interacts with Rubisco.While the low occupancy (+)-ABA binding site and weak non-competitive inhibition of catalysis may not be relevant, the high affinity site may allow ABA to act as a negative effector of Rubisco activation.

View Article: PubMed Central - PubMed

Affiliation: National Research Council of Canada, Saskatoon, Saskatchewan, Canada; Department of Chemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.

ABSTRACT
Abscisic acid ((+)-ABA) is a phytohormone involved in the modulation of developmental processes and stress responses in plants. A chemical proteomics approach using an ABA mimetic probe was combined with in vitro assays, isothermal titration calorimetry (ITC), x-ray crystallography and in silico modelling to identify putative (+)-ABA binding-proteins in crude extracts of Arabidopsis thaliana. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was identified as a putative ABA-binding protein. Radiolabelled-binding assays yielded a Kd of 47 nM for (+)-ABA binding to spinach Rubisco, which was validated by ITC, and found to be similar to reported and experimentally derived values for the native ribulose-1,5-bisphosphate (RuBP) substrate. Functionally, (+)-ABA caused only weak inhibition of Rubisco catalytic activity (Ki of 2.1 mM), but more potent inhibition of Rubisco activation (Ki of ~ 130 μM). Comparative structural analysis of Rubisco in the presence of (+)-ABA with RuBP in the active site revealed only a putative low occupancy (+)-ABA binding site on the surface of the large subunit at a location distal from the active site. However, subtle distortions in electron density in the binding pocket and in silico docking support the possibility of a higher affinity (+)-ABA binding site in the RuBP binding pocket. Overall we conclude that (+)-ABA interacts with Rubisco. While the low occupancy (+)-ABA binding site and weak non-competitive inhibition of catalysis may not be relevant, the high affinity site may allow ABA to act as a negative effector of Rubisco activation.

No MeSH data available.


Related in: MedlinePlus

ABA binds to spinach Rubisco with high affinity.A) Saturation curve and Scatchard plot representing specific binding of [3H]-(±)-ABA, as a difference between total and non-specific binding signal. B) Competition Binding Assay. Displacement of 25 nM (±) [3H]ABA by non-radiolabeled (+) ABA (diamonds), (-) ABA (circles), PA (squares) and trans-(+) ABA (triangle) at the indicated concentrations.
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pone.0133033.g003: ABA binds to spinach Rubisco with high affinity.A) Saturation curve and Scatchard plot representing specific binding of [3H]-(±)-ABA, as a difference between total and non-specific binding signal. B) Competition Binding Assay. Displacement of 25 nM (±) [3H]ABA by non-radiolabeled (+) ABA (diamonds), (-) ABA (circles), PA (squares) and trans-(+) ABA (triangle) at the indicated concentrations.

Mentions: In light of the relevance of Rubisco to plant metabolism, and the high scores obtained in the MS analysis, the interaction of Rubisco with ABA was selected for further characterization. Using a classical radiolabelled [3H]-(+/-)-ABA binding assay, previously established and applied to other ABA-binding proteins in our lab [31], a KD of 47.0 nM was obtained (Fig 3A) for ABA binding to non-activated Rubisco, suggesting a relatively high affinity interaction. This value is similar, to that obtained previously for RuBP binding to non-activated Rubisco by radiolabelled binding assay (KD = 21 nM) [13]. Binding specificity was then assessed by challenging the [3H] (±)-ABA binding with non-radiolabeled (+)-ABA, (-)-ABA, phaseic acid (PA) and trans- (+)-ABA. (-)-ABA and (+)-ABA were equally effective as competitors, whereas PA and trans-(+)-ABA were ineffective as competitors (Fig 3B). Together these data validate the initial pull-down interaction and suggest a strong and selective binding interaction between ABA and activated Rubisco.


Identification of Interactions between Abscisic Acid and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase.

Galka MM, Rajagopalan N, Buhrow LM, Nelson KM, Switala J, Cutler AJ, Palmer DR, Loewen PC, Abrams SR, Loewen MC - PLoS ONE (2015)

ABA binds to spinach Rubisco with high affinity.A) Saturation curve and Scatchard plot representing specific binding of [3H]-(±)-ABA, as a difference between total and non-specific binding signal. B) Competition Binding Assay. Displacement of 25 nM (±) [3H]ABA by non-radiolabeled (+) ABA (diamonds), (-) ABA (circles), PA (squares) and trans-(+) ABA (triangle) at the indicated concentrations.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4510133&req=5

pone.0133033.g003: ABA binds to spinach Rubisco with high affinity.A) Saturation curve and Scatchard plot representing specific binding of [3H]-(±)-ABA, as a difference between total and non-specific binding signal. B) Competition Binding Assay. Displacement of 25 nM (±) [3H]ABA by non-radiolabeled (+) ABA (diamonds), (-) ABA (circles), PA (squares) and trans-(+) ABA (triangle) at the indicated concentrations.
Mentions: In light of the relevance of Rubisco to plant metabolism, and the high scores obtained in the MS analysis, the interaction of Rubisco with ABA was selected for further characterization. Using a classical radiolabelled [3H]-(+/-)-ABA binding assay, previously established and applied to other ABA-binding proteins in our lab [31], a KD of 47.0 nM was obtained (Fig 3A) for ABA binding to non-activated Rubisco, suggesting a relatively high affinity interaction. This value is similar, to that obtained previously for RuBP binding to non-activated Rubisco by radiolabelled binding assay (KD = 21 nM) [13]. Binding specificity was then assessed by challenging the [3H] (±)-ABA binding with non-radiolabeled (+)-ABA, (-)-ABA, phaseic acid (PA) and trans- (+)-ABA. (-)-ABA and (+)-ABA were equally effective as competitors, whereas PA and trans-(+)-ABA were ineffective as competitors (Fig 3B). Together these data validate the initial pull-down interaction and suggest a strong and selective binding interaction between ABA and activated Rubisco.

Bottom Line: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was identified as a putative ABA-binding protein.Overall we conclude that (+)-ABA interacts with Rubisco.While the low occupancy (+)-ABA binding site and weak non-competitive inhibition of catalysis may not be relevant, the high affinity site may allow ABA to act as a negative effector of Rubisco activation.

View Article: PubMed Central - PubMed

Affiliation: National Research Council of Canada, Saskatoon, Saskatchewan, Canada; Department of Chemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.

ABSTRACT
Abscisic acid ((+)-ABA) is a phytohormone involved in the modulation of developmental processes and stress responses in plants. A chemical proteomics approach using an ABA mimetic probe was combined with in vitro assays, isothermal titration calorimetry (ITC), x-ray crystallography and in silico modelling to identify putative (+)-ABA binding-proteins in crude extracts of Arabidopsis thaliana. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was identified as a putative ABA-binding protein. Radiolabelled-binding assays yielded a Kd of 47 nM for (+)-ABA binding to spinach Rubisco, which was validated by ITC, and found to be similar to reported and experimentally derived values for the native ribulose-1,5-bisphosphate (RuBP) substrate. Functionally, (+)-ABA caused only weak inhibition of Rubisco catalytic activity (Ki of 2.1 mM), but more potent inhibition of Rubisco activation (Ki of ~ 130 μM). Comparative structural analysis of Rubisco in the presence of (+)-ABA with RuBP in the active site revealed only a putative low occupancy (+)-ABA binding site on the surface of the large subunit at a location distal from the active site. However, subtle distortions in electron density in the binding pocket and in silico docking support the possibility of a higher affinity (+)-ABA binding site in the RuBP binding pocket. Overall we conclude that (+)-ABA interacts with Rubisco. While the low occupancy (+)-ABA binding site and weak non-competitive inhibition of catalysis may not be relevant, the high affinity site may allow ABA to act as a negative effector of Rubisco activation.

No MeSH data available.


Related in: MedlinePlus