Figure 1.
Structure and biochemical characterizations of (−)-ABA.
Structures of (+)-ABA (A) and (−)-ABA (B). (C) (−)-ABA mediated inhibition of HAB1 phosphatase activity by PYLs. The concentration for each PYLs protein was 5.0 µM and for HAB1 was 3.0 µM. All experiments were repeated three times (n = 3) and error bars represented s.d. The condition measuring the phosphatase activity of HAB1 was same below unless noted.
Table 1.
Data collection and refinement statistics of PYLs and complexes.*
Figure 2.
Structural characterizations and bioactivity of PYL9-(+)-ABA.
(A) Two protomers of PYL9-(+)-ABA in each asymmetric unit. 2Fo−Fc electron density map of (+)-ABA at 1.0σ. (B) One protomer of PYL9-(+)-ABA with five cysteine residues (green) and a disulphide formed between C34 and C159. (C) The monomeric state of PYL9 in solution was confirmed by the sedimentation velocity. The sample purity for sedimentation velocity experiments was detected by SDS-PAGE and then Coomassie Brilliant Blue staining in the subplot. (D) The C159 was the key residue in disulphide bond, while the C29 competed with the C34 to form the disulphide bond. PYL9 and its mutants were under different oxidation-reduction conditions for SDS-PAGE. 1% βme or 1% βme +100 mM DTT was used as reducing agents to disrupt the disulphide bond. Only C159S mutant was not affected by the reducing agents (black arrow).
Figure 3.
Characterizations of PYL5 and PYL3-(−)-ABA.
(A). Chain A together with L2 of chain B and L4 of chain C displayed a whole protomer of apo-PYL5 (also seen Fig. S4C). (B) Four PYL3-(−)-ABA molecules in an asymmetric unit, organized as two trans-dimers. (C) Close-up view of (−)-ABA (yellow) in the binding pocket. 2Fo − Fc electron density map of (−)-ABA at 1.0σ. (D) Superposition of PYL3-(−)-ABA and PYL3-(+)-ABA (PDB: 4DSC). There were partially rotation and shift between the rings in both ABA. (−)-ABA was constrained in a hydrophobic cavity with little flexibility in PYL3. (E) The binding affinity of (−)-ABA to PYL3, assessed by ITC assays, was less than that of (+)-ABA or (±)-ABA, (also seen Fig. S5D,E). (F) PP2C activity (upper panel) and GST-mediated pulldown of PYL3 mutants protein in the presence of (−)-ABA (lower panel). GST-HAB1 and PYL3, highlighted by red arrows, were visualized by Coomassie Brilliant Blue staining.
Figure 4.
The stereospecificity of PYLs to (+)-ABA or (−)-ABA.
(A) The different inhibitory efficiency of (−)-ABA by PYL3, PYL5 or PYL9. The concentrations of PYLs, HAB1 and (−)-ABA were 1 µM, 0.5 µM and 10 µM, respectively. (B) Superposition of apo-PYL5, PYL3-(−)-ABA and PYL9-(+)-ABA indicated that the major variant residues underlain the favour of PYL binding (−)-ABA. Two bulk side chains of I112 and L165 in PYL9 seriously collided to 7′ and 8′ methyl groups of (−)-ABA, respectively. The stereo constraints were vanished in PYL5 because of two corresponding small side chains (also seen Fig.S1). (C). The mutation V66I in PYL9 would give a strong coordination with 8′ and 9′ methyl groups in (−)-ABA through a strong hydrophobic network with surrounding residue V85. (D) PYL9 and PYL3 mutants were engineered to gain and cripple the binding of (−)-ABA, respectively.