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Structural plasticity of D3–D14 ubiquitin ligase in strigolactone signalling

Naturevolume 563pages652656 (2018) | Download Citation

Abstract

The strigolactones, a class of plant hormones, regulate many aspects of plant physiology. In the inhibition of shoot branching, the α/β hydrolase D14—which metabolizes strigolactone—interacts with the F-box protein D3 to ubiquitinate and degrade the transcription repressor D53. Despite the fact that multiple modes of interaction between D14 and strigolactone have recently been determined, how the hydrolase functions with D3 to mediate hormone-dependent D53 ubiquitination remains unknown. Here we show that D3 has a C-terminal α-helix that can switch between two conformational states. The engaged form of this α-helix facilitates the binding of D3 and D14 with a hydrolysed strigolactone intermediate, whereas the dislodged form can recognize unmodified D14 in an open conformation and inhibits its enzymatic activity. The D3 C-terminal α-helix enables D14 to recruit D53 in a strigolactone-dependent manner, which in turn activates the hydrolase. By revealing the structural plasticity of the SCFD3–D14 ubiquitin ligase, our results suggest a mechanism by which the E3 coordinates strigolactone signalling and metabolism.

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Data availability

Structural coordinates and structural factors have been deposited in the RCSB Protein Data Bank under accession numbers 6BRO, 6BRP, 6BRQ and 6BRT. Uncropped gels and blots are available in the Supplementary Information. All other data are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank the beamline staff at ALS and APS for help with data collection, J. Nemhauser for Arabidopsis seeds, and members of the Zheng and Wenqing Xu laboratories for discussion and help. We thank L. Sheard for her early support of the project. This research is supported by the Howard Hughes Medical Institute (N.Z.), the Gatsby Charitable Foundation (GAT3272C, O.L.) and the European Research Council (294514-EnCoDe, O.L.).

Reviewer information

Nature thanks P. McCourt and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

    • Nitzan Shabek

    Present address: Department of Plant Biology, University of California, Davis, Davis, CA, USA

Affiliations

  1. Department of Pharmacology, University of Washington, Seattle, WA, USA

    • Nitzan Shabek
    • , Haibin Mao
    • , Thomas R. Hinds
    •  & Ning Zheng
  2. Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA

    • Nitzan Shabek
    • , Haibin Mao
    •  & Ning Zheng
  3. The Sainsbury Laboratory, University of Cambridge, Cambridge, UK

    • Fabrizio Ticchiarelli
    •  & Ottoline Leyser

Authors

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  6. Search for Ning Zheng in:

Contributions

N.S., N.Z. and O.L. conceived—and N.S. conducted—the protein purification and crystallization experiments. N.S. and N.Z. determined and analysed the structures. N.S. and T.R.H. conceived and conducted the AlphaScreen and BLI experiments. N.S., H.M. and F.T. conducted mutational studies and analyses. N.S. and N.Z. wrote the manuscript with the help of all other co-authors.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Ning Zheng.

Extended data figures and tables

  1. Extended Data Fig. 1 Conservation and conformation of D3 C-terminal α-helix.

    a, Sequence alignment of the C-terminal regions of 14 orthologues of MAX2 or D3. Highly conserved residues are coloured in blue. b, Electrostatic-potential surface map of D3 with CTH shown in cartoon representation (orange). The C terminus aspartic acid residue (Asp720) is anchored to a positively charged pocket. c, Close-up view of the D3 extreme C-terminal residue (Asp720) and its interacting residues in D3 and ASK1. d, Electron densities of the D3-CTH region in two different crystal forms, adopting either a regular helical conformation (left) or an extended conformation (right).

  2. Extended Data Fig. 2 Sequence alignment and analysis of selected orthologues of D3 or MAX2.

    Orthologues of D3 or MAX2 are selected and aligned from rice (O. sativa) (accession XP_015643693), A. thaliana (NP_565979), castor (Ricinus communis) (XP_002528551), poplar (Populus trichocarpa) (XP_002320412), grapevine (Vitis vinifera) (XP_010657042), cucumber (Cucumis sativus) (XP_004137031), monkey flower (Erythranthe guttata) (XP_012832933), tobacco (Nicotiana sylvestris) (XP_009757168), medicago (Medicago truncatula) (XP_003607592), pea (P. sativum) (ABD67495), soybean (Glycine max) (XP_003540983), maize (Zea mays) (XP_020394883), sorghum (Sorghum bicolor) (XP_002436499) and moss (Physcomitrella patens) (XP_024400746). The non-conserved region designed to be truncated by TEV cleavage during recombinant D3 purification is underlined in green.

  3. Extended Data Fig. 3 Comparison of D3–ASK1 structures.

    a, Top view of ASK1–D3 crystal structure (orange) based on PDB 5HYW. Red arrows indicate a gap in the polypeptide model. Note that PDB 5HYW has a polypeptide register error ranging from amino acid 373 to 473 before the gap. b, Superposition of ASK1–D3 determined in this study (light blue) with PDB 5HYW. The region truncated by design ranges from N474 to L516, which are indicated by red arrows. c, Superposition of all three crystal forms of ASK1–D3 determined in the current study. d, Limited trypsin digestion assay of ASK1–D3 and ASK1–D3(ΔCTH). Proteins were resolved by SDS–PAGE followed by Coomassie blue stain, focusing on D3 C-terminal domain. The experiment was repeated three times.

  4. Extended Data Fig. 4 Established binding between D3 and D14.

    a, AlphaScreen assay measuring direct interaction between GST–D14 and His–D3 in response to increasing amounts of GR24 (mean ± s.d. of biological triplicates). b, The binding interface between CLIM-bound D14 (magenta) and the LRR domain of D3 (blue) (PDB 5HZG). The last four LRRs are labelled, and D3-CTH in LRR20 is coloured in orange.

  5. Extended Data Fig. 5 Structural analysis of D3-CTH–D14–GR24 complex.

    a, Packing of two D14 molecules that are N-terminally fused with D3-CTH. The D3-CTH region in chain A is omitted. The GR24 D ring (sticks) is shown together with the surround 2Fo − Fc electron-density map calculated before the compound modelled in and contoured at 0.8σ. b, A close-up view of the GR24 D ring (sticks, green) and its electron density, calculated as in a. c, Overall structure of D14 (magenta) bound to D3-CTH (orange), with a GR24 D ring (green sticks). The GR24 hydrolysis product D-OH (cyan sticks)—revealed in the D14-D-OH structure (PDB 3WIO)—is shown on the basis of superposition analysis. d, Kinetics of YLG hydrolysis by free D14 and D14 fused to D3-CTH. Experiment repeated three times. e, Comparison of the interface that D14 (magenta and brown) makes upon binding to D3-CTH (orange) versus upon binding to ASK1–D3 (blue). The lid domain (brown) of D14 adopts open and closed conformation upon binding to D3-CTH and ASK1–D3, respectively. f, Electrostatic-potential surface map of D14 bound to D3-CTH (orange). The dashed line indicates the C-terminal region of D3 that would otherwise be free, if D3-CTH were not fused to another copy of D14 in the crystal. g, Conformational changes in the lid domain of D14, induced by D3-CTH binding, as revealed by superposition analysis between D3-CTH-bound (magenta) and apo D14 (grey, PDB 4IH9). Arrows indicate the rotation of the lid domain of D14, induced by D3-CTH (orange), relative to the catalytic triad shown in sticks. h, Superposition analysis of apo D14 (PDB 3W04) and D14 bound to D3-CTH, which highlights a possible allosteric pathway that connects Leu707 of D3-CTH to the catalytic triad of D14. Arrows indicate conformational changes within D14 that are induced by binding to D3-CTH.

  6. Extended Data Fig. 6 The formation of the D3–D14–D53 complex is mediated by the D2 domain of D53.

    a, Pull-down assay using recombinant ASK1–D3, His–D14, and GST-tagged N domain (D53-N), D1 domain (D53-D1) or D2 domain of D53. bd, Size-exclusion chromatography analyses of the interaction between: full-length GST–D53, D14–GR24 and ASK1–D3 (b), D14–GR24 and either ASK1–D3 or ASK1–D3(ΔCTH) (c), and D14–GR24 and D53-D2 with ASK1–D3(ΔCTH) (d). All gels were resolved by SDS–PAGE and analysed by western blot using anti-GST and anti-His antibodies (as indicated under a) or Coomassie blue staining (bd). All experiments shown in ad were repeated independently at least three times.

  7. Extended Data Fig. 7 D3-CTH facilitates the binding of the D2 domain of D53 to D14–D3.

    a, GST pull-down assay using GST–D53-D2 or the GST-tagged D2 domain of the d53 (GST–d53-D2) mutant with non-tagged D14, in the presence or absence of D3-CTH as indicated. b, AlphaScreen data showing the ability of the D3-CTH peptide (28 amino acids, D3(693–720)) to promote the interaction between D53-D2 and D14 in a dose-dependent manner; D3(693–707) (15 amino acids) and D3(708–720) (13 amino acids) peptides did not stimulate binding. DMSO (indicated as ‘no peptide’) served as control (data are mean ± s.d. of biological triplicates). c, GST pull down using recombinant GST–D53-D2 and His–D3-ASK1 in the presence of recombinant D14 wild type (WT), D14(A223E), D14(S224E) and GR24 as indicated. d, GST pull down in the presence of the D3-CTH peptide with or without GR24, and in the presence of GST–D14 wild type or GST–D14(S224E). BSA was used in the assay to prevent non-specific interactions. MG132 was added as indicated. Proteins were resolved using SDS–PAGE, and were visualized by Coomassie blue staining or western-blot using anti-GST antibodies. The D3-CTH peptide contains four amino acid mutations that were designed to disrupt the D14–D3-CTH interface: E700R, L707R, D719R and D720R. e, f, Degradation of GST–D53-D2 in the Col-0 (e) or max2-1 (f) A. thaliana cell-free extract system. GST–D53-D2 was resolved at the indicated time in the presence or absence of the wild-type D3-CTH peptide (e, top) or a mutant (MT) (e, bottom), and in the presence of D3 and either D14 wild type or the D14(S224E) mutant (f). g, Time-dependent degradation of GST–D53-D2 and GST–d53-D2 in Arabidopsis seedlings of Col-0 extracts. Proteins were resolved by SDS–PAGE, and analysed by western blot using anti-GST antibody. MG132 indicates the addition of proteasome inhibitor. h, Size-exclusion chromatography analysis of complex formation among D53-D2, ASK1–D3 and D14 in the presence of YLG. i, Kinetics of YLG hydrolysis by D14 in the presence of ASK1–D3 and D53-D2 at two concentrations. Gels were resolved by SDS–PAGE and analysed by western blot using anti-GST and anti-His antibodies as indicated under c, eg. All experiments were repeated independently at least three times.

  8. Extended Data Fig. 8 A model for strigolactone perception and signalling.

    A model of the activity cycle that underlies strigolactone-induced and SCFD3–D14-mediated D53 polyubiquitination. D3 adopts two conformational states with a structurally variable CTH (left). With a dislodged CTH, D3 binds and inhibits D14 in its open conformation, until D53 is loaded (top). D53 binding re-activates D14, which can hydrolyse strigolactones after or while D53 is polyubiquitinated. The strigolactone hydrolysis intermediate then stabilizes the closed conformation of D14, which converts D3-CTH into its engaged form. The resulting complex can ubiquitinate D14 and feed D3 back to the activity cycle (right). CLIM-bound D14 might participate in D53 polyubiquitination or in an alternative path (bottom). It remains unknown how many strigolactone molecules are hydrolysed during the polyubiquitination of each D53 molecule.

  9. Extended Data Table 1 Data collection and refinement statistics

Supplementary information

  1. Supplementary Information

    This file contains Supplementary Figure 1: Uncropped SDS-PAGE gel scans, and a Supplementary Discussion

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