Structure-Based Modulation of the Ligand Sensitivity of a Tomato Dimeric Abscisic Acid Receptor Through a Glu to Asp Mutation in the Latch Loop

Abstract

The binding of the plant phytohormone Abscisic acid (ABA) to the family of ABA receptors (PYR/PYL/RCAR) triggers plant responses to abiotic stress. Thus, the implementation of genetic or chemical strategies to modulate PYR/PYL activity might be biotechnologically relevant. We have employed the available structural information on the PYR/PYL receptors to design SlPYL1, a tomato receptor, harboring a single point mutation that displays enhanced ABA dependent and independent activity. Interestingly, crystallographic studies show that this mutation is not directly involved in ABA recognition or in the downstream phosphatase (PP2C) inhibitory interaction, rather, molecular dynamic based ensemble refinement restrained by crystallographic data indicates that it enhances the conformational variability required for receptor activation and it is involved in the stabilization of an active form of the receptor. Moreover, structural studies on this receptor have led to the identification of niacin as an ABA antagonist molecule in vivo. We have found that niacin blocks the ABA binding site by mimicking ABA receptor interactions, and the niacin interaction inhibits the biochemical activity of the receptor.

Keywords: abiotic stress; abscisic acid; plant biology; protein crystallography; signal transduction; structural biology.

FIGURE 1

Biochemical properties of SlPYL1 E151D. (A) Determination of the IC50 for inhibition of HAB1 by ABA and SlPYL1 or SlPYL1 E151D at 1:2 molar proportion of HAB1:SlPYL1. (B) HAB1 activity in the presence of molar ratios of HAB1:SlPYL1 or HAB1:SlPYL1 E151D of 1:2 and 1:4. 100% of activity is referred to 1:2 HAB1:SlPYL1wt. (C) Elution profile of SlPYL1 and SlPYL1 E151D, loading 60 (main profile), 6 or 0.6 microg of protein (inset). The positions of the molecular weight markers are indicated. (D) Thermal denaturation profiles of (left) wild-type and (right) mutant SlPYL1 E151D proteins (6 mM) in the absence or presence of increasing ABA concentrations (legend). The inflection temperature at 4 mM ABA is indicated.

FIGURE 2

The structure of the SlPYL1 E151D. (A; Right) Superposition of the structures of the apo and ABA bound SlPYL1 E151D (yellow and green, respectively) together with a detail of the residues conforming the Glu/Asp-His-Arg motif (left). The view faces the dimerization interface. The latch and gate loops are highlighted. (B) Superposition of a section of the structures of the apo and ABA bound SlPYL1 E151D (green; upper and lower panel, respectively) and SlPY1 in complex with ABA [wheat; Protein Data Bank (PDB) codes 5MOA and 5MOB, respectively]. Hydrogen bonds are represented as dashed lines.

FIGURE 3

Molecular motions of SlPYL1 E151D. (A) Overview of main chain dynamics of gate and latch in ensemble structures of the apo and ABA bound forms of SlPYL1 E151D (green) and SlPYL1 (wheat). (B) Main chain atomic root mean squared fluctuations at gate and latch of ensemble models of apo and ABA SlPYL1 E151D (solid and dashed green lines, respectively) and apo and ABA SlPYL1 (solid and dashed wheat lines, respectively). (C; Left) Side chain dynamics of residues conforming the salt bridge between Glu/Asp 151 and Arg 153 of the apo forms of SlPYL1 E151D (green) and SlPYL1 (wheat). (Right) Panel showing the corresponding minimum distance between Asp (green) or Glu 151 (wheat) carboxylate oxygen atoms and Arg 153 guanidinium nitrogen atoms distribution.

FIGURE 4

The SlPY1 E151D Niacin complex. (A) The network of hydrogen bond interactions with niacin in the complex with SlPYL1 E151D. (B) The overlay of the niacin molecules from the complex with SlPYL1 E151D onto the CsPYL1-ABA-HAB1 complex, residues are labeled according to SlPYL1 sequence. Red dashed symbols highlight hypothetical short unfavorable clashes between niacin and residues of the gate loop in closed conformation. (C) The network of hydrogen bond interactions with niacin in the complex with SLPYL1.

FIGURE 5

ABA-mediated inhibition of seedling establishment is counteracted by increasing concentrations of nicotinic acid. (A) Approximately 25 seeds (two replicates per experiment) of Arabidopsis thaliana Col-0 seeds were sown on 24-multiwell plates lacking (MOCK control) or supplemented with 0.125 μM ABA. Increasing concentrations (0.5, 2.5, and 5 μM) of nicotinic acid were added in those wells supplemented with 0.125 μM ABA. Seedling establishment and early seedling growth was scored at 5 and 9 days after sowing. (B) Root length was scored at 5 days after sowing. *p < 0.05. (C) ABA-antagonist activity of nicotinic acid. Increasing concentrations (0.25, 2.5, and 5 μM) of nicotinic acid were added to the reaction mixture supplemented with 0.25 μM ABA.

FIGURE 6

Asp at the latch contributes to the stabilization of the closed conformation of the gate. A section of the structures of dimeric AtPYL2-ABA (left) and monomeric AtPYL9-ABA (right). Selected hydrogen bonds are highlighted as dashed lines.