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A conformational switch in the SCF-D3/MAX2 ubiquitin ligase facilitates strigolactone signalling

Abstract

Strigolactones (SLs) are a class of plant hormones that regulate numerous processes of growth and development. SL perception and signal activation involves interaction between F-box E3 ubiquitin ligase D3/MAX2 and DWARF14 (D14) α/β-hydrolase in a SL-dependent manner and targeting of D53/SMXL6/7/8 transcriptional repressors (SMXLs) for proteasome-mediated degradation. D3/MAX2 has been shown to exist in multiple conformational states in which the C-terminal helix (CTH) undergoes a closed-to-open dynamics and regulates D14 binding and SL perception. Despite the multiple modes of D3–D14 interactions found in vitro, the residues that regulate the conformational switch of D3/MAX2 CTH in targeting D53/SMXLs and the subsequent effect on SL signalling remain unclear. Here we elucidate the functional dynamics of ASK1–D3/MAX2 in SL signalling by leveraging conformational switch mutants in vitro and in plants. We report the crystal structure of a dislodged CTH of the ASK1–D3 mutant and demonstrate that disruptions in CTH plasticity via either CRISPR–Cas9 genome editing or expression of point mutation mutants result in impairment of SL signalling. We show that the conformational switch in ASK1–D3/MAX2 CTH directly regulates ubiquitin-mediated protein degradation. A dislodged conformation involved in D53/SMXLs SL-dependent recruitment and ubiquitination and an engaged conformation are required for the release of polyubiquitinated D53/SMXLs and subsequently D14 for proteasomal degradation. Finally, we uncovered an organic acid metabolite that can directly trigger the D3/MAX2 CTH conformational switch. Our findings unravel a new regulatory function of a SKP1–CUL1–F-box ubiquitin ligase in plant signalling.

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Fig. 1: Structural and functional characterization of dislodged ASK1–D3-CTH.
Fig. 2: Functional characterization of dislodged ASK1–D3-CTH mutant in systemic SL signalling.
Fig. 3: Functional characterization of D-pocket perturbed ASK1–D3 mutant in systemic SL signalling.
Fig. 4: CTH dynamics affect D53/SMXL7 ubiquitin-mediated degradation by the Ub ligase D3/MAX2.
Fig. 5: Structure–function analysis of ASK1–D3-citrate complex dynamics.
Fig. 6: CRISPR–Cas9-guided mutagenesis of CTH of MAX2 results in SL-signalling deficiency phenotypes.
Fig. 7: An updated model for SL signalling.

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Data availability

The atomic coordinates and all related data of ASK1–D3 mutant structure were deposited in the Protein Data Bank with accession code 7SA1 (PDB:7SA1). All other materials are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

N.S. is supported by National Science Foundation, NSF-CAREER (Award No. 2047396) and NSF-EAGER (Award No. 2028283). We thank the beamline staff for providing technical assistance at the Advanced Light Source (U.S. Department of Energy Office of Science User Facility under Contract No. DE-AC02-05CH11231, supported in part by the ALS-ENABLE program funded by the National Institutes of Health, National Institute of General Medical Sciences, grant P30 GM124169-01). A.B.'s laboratory is supported by National Science Foundation, NSF BTT EAGER (Award No. 1844705). L.T. is supported by BARD, the United States–Israel Binational Agricultural Research and Development Fund, Vaadia-BARD Postdoctoral Fellowship Award FI-559-2017.

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Contributions

L.T. and N.S. conceived and designed the experiments. N.S. and L.T. conducted the protein purification, biochemical and crystallization experiments with the help of A.Y. and M.P. Structural and functional analyses were determined and performed by N.S., L.T. and M.P. N.S. and M.P. designed and performed molecular dynamics simulations. N.S. and L.T. designed, and L.T. generated the transgenic lines including phenotype characterizations. L.T., M.R. and A.B. designed and L.T. generated CRISPR–Cas9 Arabidopsis lines. N.S. and L.T. wrote the manuscript with the help from M.P., M.R. and A.B.

Corresponding author

Correspondence to Nitzan Shabek.

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Competing interests

N.S. has an equity interest in Oerth Bio and serves on the company’s Scientific Advisory Board. The work and data submitted here have no competing interests, nor other interests that might be perceived to influence the results and/or discussion reported in this paper. The remaining authors declare no competing interests.

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Nature Plants thanks Tom Bennett, Shinjiro Yamaguchi and the other, anonymous, reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Molecular dynamics simulation of ASK1-D3D720K and functionality analyses.

a, Molecular dynamics simulation of ASK1-D3 and ASK1-D3D720K. Root mean square deviation (RMSD, upper panels) and RMS fluctuation (RMSF, lower panels) of all atoms plotted as a function of time for ASK-D3 (brown) and ASK1-D3D720K (cyan) (left panels) and for the CTH of ASK-D3 and ASK1-D3D720K (right panels). b, Conformational clusters over time plotted with the cut-off of 0.25 nm. c, Pulldown of GST-D14 and His-D3 or His-D3D720K in the presence or absence of the synthetic SL, GR24. Proteins were resolved by SDS-PAGE and visualized via Western blot with anti-His and anti-GST antibodies. Asterisk denotes non-specific protein band. d, D14-YLG hydrolysis assay in the presence of either ASK1-D3, or ASK1-D3D720K. Colored lines represent non-linear regression curved fit based on duplications of the raw data points (shown in dots).

Source data

Extended Data Fig. 2 max2 complementation experiments.

a, Top panel shows 5 week old plants, SL deficiency phenotypes include changes in rosettes size and leaf shape. Bottom panel shows 8 week old plants, an SL deficiency phenotype of excessive branching is shown. Transgenic plants are in max2 background. b, Mean number of axillary rosettes branches (± SE). (WT (n = 7), max2 (n = 7), pUBQ:MAX2 (n = 8) and pUBQ:MAX2D693K (n = 11), One-way ANOVA and post hoc Tukey test, P < 0.001). Transgenic plants are in max2 background.

Source data

Extended Data Fig. 3 D-pocket perturbed MAX2 plants.

5-week-old plants show SL deficiency phenotypes including changes in rosettes size and leaf shape (upper panel). 8-week-old plants exhibit SL deficiency phenotype of excessive branching (lower panel). Transgenic plants are in WT Col-0 background.

Extended Data Fig. 4 Ubiquitination and time scale degradation of GST-SMXL7D2.

a, Representative Western blot gel of a polyubiquitinated D53D2 levels measurement using anti-GST (α-GST), anti-Ubiquitin (α-Ubiquitin) and anti-His (a-His) antibodies in the presence of max2 total protein extract and His-D3 or His-D3D720K. b, Time scale of cell-free degradation of GST-SMXL7D2 in comparison to a sample incubated with protease inhibitor MG132. Loading control (LD) is shown by Ponceau stain.

Source data

Extended Data Fig. 5 functional analysis of ASK1-D3-citrate complex dynamics.

a, D14-YLG hydrolysis assay in the presence of either ASK1-D3, or ASK1-D3D720K, and succinate (Suc, left) or citrate (Cit, right) as indicated. Colored lines represent non-linear regression curved fit based on duplications of the raw data points (shown in dots). b, Molecular docking, and fitting scores of the different ligands from ASK1-D3D720K crystallization conditions are shown in the table with DG values. Closeup view of the 2Fo-Fc (1.0 s, left panel) map and Fo-Fc (3 s, right panel) show fitting of citrate into Fo-Fc electron density and its interaction with residues in the D-Pocket. 2-D interaction plot was generated using LigPlot+ and shows citrate interaction network with the D-pocket (black dash lines represent hydrogen bonds, arches represent Van der Waals interactions). c, Representative Western blot gel of a polyubiquitinated D53D2 levels measurement using anti-GST (α-GST), anti-Ubiquitin (α -Ubiquitin) and anti-His (α -His) antibodies, in the presence of WT total protein extract with citrate or succinate d, Cell-free degradation of GST-AUX/IAA17D2 detected by Western blot using anti-GST (α -GST) antibody. Numbers under the blots are proportions of protein remained that were quantified and compared to T = 0. The experiment was repeated three times.

Source data

Extended Data Fig. 6 Genome edited MAX2 Arabidopsis lines.

a, Schematic representation of MAX2 protein variants generated by CRISPR/Cas9. MAX2 gene diagram is shown with focus on MAX2 coding sequence for the CTH amino acids. gRNAs are colored in grey with PAM sequence colored yellow. All WT sequences are shown in blue. DNA deletions as well as protein sequence changes are shown in red. b, Mean leaf blade length and width in the 7th leaf of WT, max2, CRISPR/CAS9 edited MAX2 mutants and kai2. measured at proliferative arrest (± SE). WT (n = 15), max2 (n = 14), MAX2∆CTH (n = 23), MAX2NH (n = 25), MAX2KSLTET (n = 29), MAX2NID (n = 23) and kai2 (n = 21); bars with the same letter are not significantly different from one another (One-way ANOVA and post hoc Tukey, P < 0.05). c, Mean of primary floral stem thickness of WT, max2 and MAX2 genome edited CRISPR/CAS9 lines. (± SE). n = WT (n = 17), max2 (n = 10), MAX2∆CTH (n = 28), MAX2NH (n = 27), MAX2KSLTET (n = 28) and MAX2NID (n = 20); bars with the same letter are not significantly different from one another (One-way ANOVA and post hoc Tukey, P < 0.05). d-e, Structural simulations of AtMAX2 mutants reveal salt bridges between H691 (b) or K690 (c) of CTH and E149 of ASK1.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2 and Table 1

Reporting Summary

Source data

Source Data Fig. 1

Unprocessed western blots and gels.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Unprocessed western blots.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Unprocessed western blots and gels.

Source Data Fig. 5

Statistical source data.

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Statistical source data.

Source Data Extended Data Fig. 1

Unprocessed western blots.

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Statistical source data.

Source Data Extended Data Fig. 4

Unprocessed western blots.

Source Data Extended Data Fig. 5

Unprocessed western blots.

Source Data Extended Data Fig. 6

Statistical source data.

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Tal, L., Palayam, M., Ron, M. et al. A conformational switch in the SCF-D3/MAX2 ubiquitin ligase facilitates strigolactone signalling. Nat. Plants 8, 561–573 (2022). https://doi.org/10.1038/s41477-022-01145-7

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