Abstract
PYL10 is a monomeric abscisic acid (ABA) receptor that inhibits protein phosphatase 2C (PP2C) activity in Arabidopsis thaliana. Previous studies reported that the PP2C phosphatase inhibition by PYL10 was ABA-independent. Here, systematic PYL10 biochemical studies demonstrated that PYL10 activity was ABA-dependent, and the previously reported studies was interfered by the presence of BSA in the commercial kit. To investigate dynamic mechanism of how ABA binding to PYL10 induces PP2C phosphatase inhibiting activity, solution NMR relaxation analysis of apo-PYL10 and PYL10/ABA were conducted following backbone resonance assignments. Reduced spectrum density mapping of the backbone relaxation data revealed that PYL10 was more flexible in ABA bound form than apo-PYL10, indicating an increased conformational entropy upon ligand binding. Moreover, to illustrate conformation exchanges of PYL10 upon ABA binding, NMR line shape analysis was performed with increasing concentrations of ABA, and the results indicated that PYL10 backbone conformational changes occur at different time scales.
Highlights
The apo- form of PYL10 was reported in an open conformation, with the CL2 region pointing away from the entrance to the Abscisic acid (ABA) binding pocket[16]
PP2C inhibition activity of PYL10 is enhanced by ABA as well as BSA
During PP2C phosphatase activity experiments in our lab we used home-made buffers instead of those included in commercially available kinase kits (Ser/Thr phosphatase Assay System, Promega, WI, USA), and noticed that the PP2C inhibition activity of PYL10 was disrupted by BSA that is normally included to avoid non-specific interactions (Fig. 1)
Summary
The apo- form of PYL10 was reported in an open conformation, with the CL2 region pointing away from the entrance to the ABA binding pocket[16]. We serendipitously discovered that the PP2C inhibition activity of monomeric PYLs was enhanced by ABA, this activity was disrupted by BSA in the commercial kinase assay kit. Solution NMR relaxation measurements and consequent dynamic analysis results demonstrated that most residues of PYL10 became more flexible upon ABA binding. We propose that ABA binding to PYL10 leads to elevated conformational entropy (Δ S) and decreased Gibbs free energy (Δ G). The ABA binding to PYL10 could enhance the PP2C phosphatase inhibition activities of PYL10
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