Structural Basis of Karrikin and Non-natural Strigolactone Perception in Physcomitrella patens

Abstract

In plants, strigolactones are perceived by the dual receptor-hydrolase DWARF14 (D14). D14 belongs to the superfamily of α/β hydrolases and is structurally similar to the karrikin receptor KARRIKIN INSENSITIVE 2 (KAI2). The moss Physcomitrella patens is an ideal model system for studying this receptor family, because it includes 11 highly related family members with unknown ligand specificity. We present the crystal structures of three Physcomitrella D14/KAI2-like proteins and describe a loop-based mechanism that leads to a permanent widening of the hydrophobic substrate gorge. We have identified protein clades that specifically perceive the karrikin KAR₁ and the non-natural strigolactone isomer (-)-5-deoxystrigol in a highly stereoselective manner.

Keywords: Physcomitrella patens; karrikin; strigolactone; α/β hydrolases.

Figure 1.. Different PpKAI2-like Proteins Feature Different Ligand Binding Site Architectures

(A) Superimposition of PpKAI2-like C, E, and H showing the residues involved in the formation of the hydrophobic substrate tunnel. (B) Multiple/sequence alignment of PpKAI2-like proteins highlighting the amino acids shown in (A). (C) Clipped surface view of different PpKAI2-like proteins showing their tunnel diameters. (D) Interaction between helices αE and αF. F_O-F_C omit maps for the loops are shown as gray wire.

Figure 2.. Manipulation of the Loop-Based Interaction between Helices αE and αF Weakens and Abolishes (±)-GR24 Binding

(A–C) Isothermal titration calorimetry of (±)-GR24 with PpKAI2-like E wild-type (A) and mutant versions S166A (B) and S166D S170D (C). (D) Isothermal titration calorimetry of (±)-GR24 with PpKAI2-like C. (E) Comparison of flexibility of structural segments contributing to the lid structure of PpKAI2, colored according to atomic B factors.

Figure 3.. PpKAI2-like C, D, and E Are Highly Stereoselective for (−)-5-deoxystrigol, and PpKAI2-like H, K, and L Bind the Karrikin KAR₁

(A) Differential scanning fluorimetry with 4 stereoisomers of strigolactones and PpKAI2-like proteins. 2(B) Microdialysis assay to analyze KAR₁ binding by different PpKAI2-like proteins.

Figure 4.. Complementation Studies of Arabidopsis kai2 and d14 Mutant Lines Using PpKAI2L Genes

(A and B) Hypocotyl growth phenotypes (A) and KAR₁ responses (B) of Arabidopsis kai2 complemented with PpKAI2-like-C, E, and H. Wild-type KAI2 and KAI2 S95A were used as positive and negative controls, respectively. Seedlings were grown on agar plates containing DMSO (representative phenotypes are shown in A) and 1 and 10 μM KAR₁ under short-day conditions for 9 days. Scale bars represent 5 mm. Data are means ± SD (n = 7–9). Means with asterisks indicate significant inhibitions from mock-treated seedlings in each line (Tukey-Kramer, p < 0.05). (C) Gene expression analysis of PpKAI2-like C, E, and H in the transgenic lines used in (A) and (B) by semi-qRT-PCR. Actin was used as an internal control. (D and E) Shoot-branching phenotypes (D) of Arabidopsis d14 complemented with PpKAI2-like C, E, and H. Plants were grown for 45 days. The number of axillary branches (>5 mm) per plant is shown (E) as means ± SD (n = 13–14). Means with different letters indicate significant differences (Steel-Dwass, p < 0.05). (F) Gene expression analysis of PpKAI2-like C, E, and H in the transgenic lines used in (D) and (E) by semi-qRT-PCR.