Conformational Dynamics of the D53-D3-D14 Complex in Strigolactone Signaling

Strigolactones (SLs) play fundamental roles in regulating plant architecture, which is a major factor determining crop yield. The perception and signal transduction of SLs require the formation of a complex containing the receptor DWARF14 (D14), an F-box protein D3 and a transcriptional regulator D53 in an SL-dependent manner. Structural and biochemical analyses of D14 and its orthologs DAD2 and AtD14, D3 and the complexes of ASK1-D3-AtD14 and D3CTH-D14 have made great contributions to understanding the mechanisms of SL perception. However, structural analyses of D53 and the D53-D3-D14 holo-complex are challenging, and the biochemical mechanism underlying the complex assembly remains poorly understood. Here, we found that apo-D53 was rather flexible and reconstituted the holo-complex containing D53, S-phase kinase-associated protein 1 (SKP1), D3 and D14 with rac-GR24. The cryo-electron microscopy (cryo-EM) structure of SKP1-D3-D14 in the presence of D53 was analyzed and superimposed on the crystal structure of ASK1-D3-AtD14 without D53. No large conformational rearrangement was observed, but a 9Å rotation appeared between D14 and AtD14. Using hydrogen-deuterium exchange monitored by mass spectrometry, we analyzed dynamic motifs of D14, D3 and D53 in the D53-SKP1-D3-D14 complex assembly process and further identified two potential interfaces in D53 that are located in the N and D2 domains, respectively. Together, our results uncovered the dynamic conformational changes and built a model of the holo-complex D53-SKP1-D3-D14, offering valuable information for the biochemical and genetic mechanisms of SL perception and signal transduction.