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.

独脚金内酯 (SLs) 是一类调节许多生长和发育过程的植物激素。SL 感知和信号激活涉及 F-box E3 泛素连接酶 D3/MAX2 和 DWARF14 (D14) α/β-水解酶之间以 SL 依赖性方式相互作用和靶向蛋白酶体的 D53/SMXL6/7/8 转录抑制因子 (SMXLs)介导的降解。D3/MAX2 已被证明以多种构象状态存在，其中 C 末端螺旋 (CTH) 经历从封闭到开放的动力学并调节 D14 结合和 SL 感知。尽管在体外发现了多种 D3-D14 相互作用模式，但在靶向 D53/SMXL 中调节 D3/MAX2 CTH 构象转换的残基以及随后对 SL 信号传导的影响仍不清楚。在这里，我们通过在体外和植物中利用构象转换突变体来阐明 ASK1-D3/MAX2 在 SL 信号传导中的功能动力学。我们报告了 ASK1-D3 突变体脱落的 CTH 的晶体结构，并证明通过 CRISPR-Cas9 基因组编辑或点突变突变体的表达破坏 CTH 可塑性会导致 SL 信号传导受损。我们表明 ASK1–D3/MAX2 CTH 中的构象转换直接调节泛素介导的蛋白质降解。涉及 D53/SMXLs SL 依赖性募集和泛素化的构象和接合构象是释放多泛素化 D53/SMXLs 和随后释放 D14 用于蛋白酶体降解所必需的。最后，我们发现了一种可以直接触发 D3/MAX2 CTH 构象转换的有机酸代谢物。