The HAB1 PP2C is inhibited by ABA-dependent PYL10 interaction

Abscisic acid (ABA) is an essential phytohormone that contributes to growth, development, and stress responses to drought, salinity or pathogens in plants1,2. The PYR1/PYL/RCAR family (hereafter referred to as PYLs for simplicity) contains 14 proteins (PYR1 and PYL1-13) in Arabidopsis thaliana that have been identified as soluble ABA receptors and therefore play a critical role in ABA signal transduction3,4. Bioinformatics and crystallographic studies revealed that members of the PYL family are highly homologous in both sequence and structure. PYLs contain a characteristic star-related lipid transfer (START) domain5. Upon binding of ABA, PYLs undergo a conformational transition from an open to a closed state via flexible ‘gate’ and ‘latch’ loops, and the resultant ligand-bound conformation can effectively bind to the active site of downstream type 2C protein phosphatases (PP2Cs) to competitively inhibit their phosphatase activity. This subsequently disrupts the PP2C-mediated inhibition of downstream sucrose non-fermenting1-related subfamily 2 (SnRK2) kinases6,7,8,9,10 that are then activated by autophosphorylation and can stimulate the expression of ABA-responsive genes11,12,13. Despite the high sequence and structural conservation of PYL proteins, different members display distinct differences in ABA selectivity and sensitivity14. Previous biochemical studies suggest that the 14 PYL proteins at least have two mechanism in the inhibition on PP2C: ABA-dependent and ABA-independent. Some dimeric PYLs (such as PYR1, PYL1 and PYL2) are classified as ABA-dependent, since it was reported that they could dissociate and interact with PP2C to inhibit its phosphatase activity upon ABA binding6. Some other ABA receptors were recently reported to inhibit PP2C activity in the absence of ABA15,16. Especially, the monomeric PYLs can form a stronger basal interaction with ABA than do dimeric PYLs15. However, whether the monomeric PYLs were really in the mechanism of ABA-independent still need further evidence. To investigate ABA binding, we recently solved crystal structures of PYL10 in the apo- and ABA-bound forms17. These structures were very similar, and both have a closed gate loop (CL2) (Fig. S1B), consistent with the proposed ABA-independent PP2C inhibition of PYL1016. However, another crystal structure of the apo- form of PYL10 was reported in an open conformation, with the CL2 region pointing away from the entrance to the ABA binding pocket16. Therefore, the CL2 region appears to be highly flexible and able to adopt different conformations, two of which have been captured crystallographically17. Recently, we serendipitously discovered that the PP2C inhibition activity of monomeric PYLs was enhanced by ABA, although this activity was disrupted by BSA in the commercial kinase assay kit. A systematic analysis of dimeric PYL10 and the monomeric mutants was subsequently conducted to verify the ABA dependent activity of monomeric PYLs. Additionally, to exclude conformation effects from crystal lattice packing, solution NMR experiments were performed to analyze the dynamic properties of PYL10 in the absence or presence of ABA. Solution NMR relaxation measurements and consequent dynamic analysis results demonstrated that most residues of PYL10 became more flexible upon ABA binding. We therefore propose that ABA binding to PYL10 leads to elevated conformational entropy (ΔS) and consequently decreased Gibbs free energy (ΔG). Therefore, the ABA binding to PYL10 could enhance the PP2C phosphatase inhibition activities of PYL10.