Small-molecule agonists and antagonists of F-box protein-substrate interactions in auxin perception and signaling

Abstract

The regulation of gene expression by the hormone auxin is a crucial mechanism in plant development. We have shown that the Arabidopsis F-box protein TIR1 is a receptor for auxin, and our recent structural work has revealed the molecular mechanism of auxin perception. TIR1 is the substrate receptor of the ubiquitin-ligase complex SCF(TIR1). Auxin binding enhances the interaction between TIR1 and its substrates, the Aux/IAA repressors, thereby promoting the ubiquitination and degradation of Aux/IAAs, altering the expression of hundreds of genes. TIR1 is the prototype of a new class of hormone receptor and the first example of an SCF ubiquitin-ligase modulated by a small molecule. Here, we describe the design, synthesis, and characterization of a series of auxin agonists and antagonists. We show these molecules are specific to TIR1-mediated events in Arabidopsis, and their mode of action in binding to TIR1 is confirmed by x-ray crystallographic analysis. Further, we demonstrate the utility of these probes for the analysis of TIR1-mediated auxin signaling in the moss Physcomitrella patens. Our work not only provides a useful tool for plant chemical biology but also demonstrates an example of a specific small-molecule inhibitor of F-box protein-substrate recruitment. Substrate recognition and subsequent ubiquitination by SCF-type ubiquitin ligases are central to many cellular processes in eukaryotes, and ubiquitin-ligase function is affected in several human diseases. Our work supports the idea that it may be possible to design small-molecule agents to modulate ubiquitin-ligase function therapeutically.

Fig. 1.

α-Alkyl IAAs act on TIR1-mediated auxin signaling. (A) The chemical structures of probes 1–8. (B and C) Effects of the probes on auxin-responsive gene expression. Arabidopsis DR5::GUS reporter line was treated with/without chemicals for 5 h. The induced GUS activity is expressed relative to 1 μM NAA treatment (100%). Error bars, mean +/− SD of three independent experiments. (B) Auxin activity of 1–3 (10 μM) and 4–8 (50 μM). (C) Antiauxin activity of 4–8 (light gray, 20 μM; dark gray, 50 μM). (D) The effect of probes on the Aux/IAA–TIR1 interaction. c-myc-tagged TIR1 was pulled-down using biotinylated IAA7 domain II peptide in the presence of chemicals (100 μM probes and/or 0.5 μM IAA), and recovery of TIR1-myc was monitored by Western blot analysis with anti-c-Myc antibody. (E) The effect of probes on Aux/IAA stability. The HS::AXR3NT-GUS line was incubated with chemicals (2 μM 3, 1 μM IAA, and/or 50 μM 4–8) after heat-shock induction. GUS expression was visualized by histochemical GUS staining.

Fig. 2.

The antiauxin probe 8 blocks typical auxin responses in the Arabidopsis root. (A) Probe 8 blocks auxin-induced root hair formation. Five-day-old seedlings were cultured with chemicals (0.1 μM IAA and/or 20 μM 8) for 36 h. Arrows indicate root hair. (B) Effect of 8 on auxin-induced lateral root formation. Five-day-old seedlings were cultured with chemicals for another 4 days. Left, untreated root; Center, treated with 0.2 μM 2,4-D; Right, treated with 0.2 μM 2,4-D and 20 μM 8. Scale bar, 10 mm. (C) Effects of 8 on root gravitropic response. Five-day-old seedlings (n = 15) were grown in the dark for 2 days after rotating seedling 135° angle along their growth axis. The arrows indicate the vector of gravity before (i) and after (ii) the start of gravistimulation. The root angles were plotted on circular histograms at 20° intervals. Assays were performed in duplicate. (D) Root tip grown for 5 days in the presence of 8 and NPA at the concentrations indicated. Lugol staining was used to show starch granules in columella cells.

Fig. 3.

The effects of probe 8 on the growth and development of Arabidopsis. (A–C) The effects of 8 on the elongation of hypocotyl and root in wild-type and auxin overproducing yucca mutants. The seedlings were grown for 6 days in the presence of chemicals. Five μM NPA and 50 nM IAA were used for assays unless otherwise stated. (A) Photos of Arabidopsis seedlings grown in the presence or absence of chemicals (a–f, wild-type; g–i, yucca mutant). Plants grown with 8 (b, c, and h), with NPA (d and i), or with IAA and/or 20 μM 8 (e and f). (B and C) Root and hypocotyl length was measured after cultivating with 8 and with and without 50 nM IAA. Values are the mean +/− SD of two independent experiments. (D) Wild-type plants treated with 8 phenocopy auxin-insensitve mutants, axr1–3, axr1–12, and axr2–1. Wild type were grown for 14 days with or without 8. Mutants were grown for 14 days without 8. (Scale bars, 5 mm in A and 10 mm in D.)

Fig. 4.

Effects of 8 on the growth of Arabidopsis auxin mutants. Arabidopsis wild type and mutants were grown for 6 days. Upper shows the effect of increasing concentrations of 8 on root length relative to untreated control (100%). (Lower) Hypocotyl length relative to untreated control (100%). Values are the mean +/− SD of two independent experiments.

Fig. 5.

Crystal structure and molecular docking analysis of TIR1–probe complexes. (A and B) Crystal structure of TIR1–probe complexes. TIR1 is shown as silver ribbon. Probes 3, 4, and 8 are shown as blue, yellow, and green, respectively. IAA7 degron peptide (pink, surface-filled model) and IAA (red) were superimposed on the coordinates in the crystal structure of the TIR1-IAA-IAA7 complex. (C) Molecular docking of TIR1 probe. Predicted binding conformers of 3 (blue) and 4 (yellow) to TIR1 auxin-binding site. Fifty possible binding conformers were predicted by the program AutoDock. Ten representative conformers were shown based on rmsd values to the coordinates of IAA moiety in 3 and 4 in crystal structure.

Fig. 6.

The TIR1/AFB specific probe 8 blocks auxin responses of moss P. patens. (A) Effects of 8 on NAA-induced elongation of P. patens gametophores. The juvenile gametophore was incubated for 60 h with chemicals (2 μM NAA and/or 20 μM 8). Arrows indicate the elongation zone in response to NAA. (Scale bar, 10 mm.) (B) Effects of 8 and NAA on the development of chloronemata. Chloronema cells were cultured on a BCDATG medium for 10 days in the presence of 0.5 μM NAA and/or 10 μM 8. Arrows indicate caulonemata. [Scale bars, 2.5 mm (Upper) and 1 mm (Lower).]