Crystal structure of Arabidopsis DWARF14-LIKE2 (DLK2) reveals a distinct substrate binding pocket architecture

Abstract

In Arabidopsis thaliana, the Sigma factor B regulator RsbQ-like family of α/β hydrolases contains the strigolactone (SL) receptor DWARF14 (AtD14), the karrikin receptor KARRIKIN INSENSITIVE2 (AtKAI2), and DWARF14-LIKE2 (AtDLK2), a protein of unknown function. Despite very similar protein folds, AtD14 and AtKAI2 differ in size and architecture of their ligand binding pockets, influencing their substrate specificity. We present the 1.5 Å crystal structure of AtDLK2, revealing the smallest ligand binding pocket in the protein family, bordered by two unique glycine residues. We identified a gatekeeper residue in the protein's lid domain and present a pyrrolo-quinoline-dione compound that inhibits AtDLK2's enzymatic activity.

FIGURE 1

Two glycine residues border a smaller substrate binding pocket in AtDLK2. (a–c) AtDLK2 has a smaller ligand binding pocket than AtD14 or AtKAI2. (d) Superimposition of the lid domains of AtDLK2, AtD14, and AtKAI2, showing that the glycine residues G154 and G201 in AtDLK2 lead to shifts of helices αT1 and αT4, pushing residues I153 and F200 into the substrate binding pocket, restraining its size. (e–g) Visualization of the substrate binding pockets of AtDLK2, AtD14, and AtKAI2, respectively. Surface is colored by hydrophobicity, with blue as hydrophilic and gray as hydrophobic. (h–j) Distances between pocket wall forming phenylalanines, demonstrating that the shift of F200 in AtDLK2 leads to a narrower pocket diameter compared with AtD14 and AtKAI2.

FIGURE 2

Phe167 partially blocks the entrance into the substrate binding pocket of AtDLK2. The 2mF _o − DF _c electron density map of the Phe167 side chain is contoured at 1 σ and shown in blue.

FIGURE 3

Thermal stability of (a) AtDLK2 wt, (b) AtDLK2 F167L, (c) AtDLK2 F167A, and (d) AtD14 in the presence of rac‐GR24. Proteins were heat‐denatured in triplicates in the presence of Sypro Orange dye using a linear 25–95°C gradient at a rate of 1°C per minute.

FIGURE 4

4,4,6‐Trimethyl‐6‐phenyl‐5,6‐dihydro‐4H‐pyrrolo[3,2,1‐ij]quinoline‐1,2‐dione (1) inhibits AtDLK2 activity. (a) Enzymatic activity was measured in triplicates as absorbance of para‐nitrophenol at 410 nm as result of enzymatic activity against pNP acetate. All values have been corrected for spontaneous pNP acetate hydrolysis. Error bars represent standard deviation. (b) Molecular docking of the inhibitor into the ligand binding pocket of AtDLK2. The protein surface is shown as electrostatic potential contoured from −12.8 kT e⁻¹ (red) to +12.8kT e⁻¹ (blue). (c–d) Compound 1 only marginally inhibits the enzymatic activity of AtD14 or AtKAI2.