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Structural insights into partner selection for MYB and bHLH transcription factor complexes

Nature Plants volume 8, pages 1108–1117 (2022)Cite this article

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Abstract

MYB and basic helix–loop–helix (bHLH) transcription factors form complexes to regulate diverse metabolic and developmental processes in plants. However, the molecular mechanisms responsible for MYB–bHLH interaction and partner selection remain unclear. Here, we report the crystal structures of three MYB–bHLH complexes (WER–EGL3, CPC–EGL3 and MYB29–MYC3), uncovering two MYB–bHLH interaction modes. WER and CPC are R2R3- and R3-type MYBs, respectively, but interact with EGL3 through their N-terminal R3 domain in a similar mode. A single amino acid of CPC, Met49, is crucial for competition with WER to interact with EGL3. MYB29, a R2R3-type MYB transcription factor, interacts with MYC3 by its C-terminal MYC-interaction motif. The WER–EGL3 and MYB29–MYC3 binding modes are widely applied among MYB–bHLH complexes in Arabidopsis and evolve independently in plants.

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Fig. 1: Co-crystal structure of the WER–EGL3 complex.
Fig. 2: Co-crystal structure of the CPC–EGL3 complex.
Fig. 3: Functional importance of Met49 of CPC in competition with WER to bind to EGL3.
Fig. 4: Co-crystal structure of the MYB29–MYC3 complex.
Fig. 5: Two interaction modes of MYB–bHLH complexes.

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Data availability

Structural factors and coordinates have been deposited in the Protein Data Bank (PDB) under accession codes 7FDL, 7FDM, 7FDN and 7FDO for WER–EGL3, MYB29–MYC3, EGL3 and CPC–EGL3. And the structure of WER–DNA complex is available in the PDB by accession code 6KKS. Source data are provided with this paper.

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Acknowledgements

We thank W. Yang, H. Ma and X. Chen for a critical reading of the manuscript. We thank Y. Ding for help in BLI and ITC experiments. We thank the staff of beamlines BL17U1, BL18U1 and BL19U1 at the Shanghai Synchrotron Radiation Facility for assistance with data collection. We thank the staff members of the Microscale Thermophoresis System at the National Facility for Protein Science in Shanghai (NFPS), Zhangjiang Lab, Shanghai Advanced Research Institute, Chinese Academy of Science, China for providing technical support and assistance in data collection and analysis. We thank R. McKenzie from Liwen Bianji (Edanz) (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript. This work was supported by the National Natural Science Foundation of China (NSFC31930017) to A.D.

Author information

Author notes

  1. These authors contributed equally: Baihui Wang, Qiang Luo, Yingping Li.

Authors and Affiliations

  1. State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, Department of Biochemistry and Biophysics, Institute of Plant Biology, Fudan University, Shanghai, P.R. China

    Baihui Wang, Qiang Luo, Yingping Li, Kangxi Du, Zhen Wu, Tianyang Li & Aiwu Dong

  2. Institut de Biologie Moléculaire des Plantes, CNRS, Université de Strasbourg, Strasbourg, France

    Wen-Hui Shen

  3. State Key Laboratory of Genetic Engineering, Center for Evolutionary Biology, Institute of Plant Biology, Fudan University, Shanghai, P.R. China

    Chien-Hsun Huang

  4. Shanghai Public Health Clinical Center, State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Department of Biochemistry and Biophysics, Fudan University, Shanghai, P.R. China

    Jianhua Gan

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Contributions

A.D. conceived and designed the research. B.W., Q.L., Y.L., K.D., Z.W. and T.L. performed the experiments. Q.L., B.W., J.G. and C.-H.H. analysed the data. Q.L., B.W., W.-H.S., C.-H.H., J.G. and A.D. wrote the manuscript. All authors read, revised and approved the manuscript.

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Correspondence to Chien-Hsun Huang, Jianhua Gan or Aiwu Dong.

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Nature Plants thanks Shingo Nagano, Toshio Hakoshima and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Mapping the interaction domains of WER and EGL3.

a, The diagram of the full-length EGL3. b, The diagram of the full-length WER. c, Mapping the interacting domains of EGL3 and WER by Y2H assay. d, SEC experiment to confirm the interaction domains within EGL3 and WER. The WER-EGL3 complex is analyzed by SDS-PAGE, which was repeated independently twice with similar results.

Source data

Extended Data Fig. 2 The interacting interface of the MYB-bHLH complexes.

a, The electrostatic surface potentials of EGL3 in the WER-EGL3 complex. The EGL3-interacting residues of WER are highlighted as sticks. b, The electrostatic surface potentials of WER in the WER-EGL3 complex. EGL3 is shown as cartoon with the WER-interacting residues highlighted as sticks. c-d, The electrostatic surface potentials of EGL3 and CPC in the CPC-EGL3 complex, respectively. The EGL3-interacting residues of CPC are highlighted as sticks in (c). EGL3 is shown as cartoon with the CPC-interacting residues highlighted as sticks in (d). e, The electrostatic surface potentials of MYC3 in the MYB29-MYC3 complex. The MYC3-interacting residues of MYB29 are highlighted as sticks. f, The electrostatic surface potentials of MYB29 in the MYB29-MYC3 complex. MYC3 is shown as cartoon with the MYB29-interacting residues highlighted as sticks. The negatively and positively charged residues are colored in red and blue on the surface, respectively.

Extended Data Fig. 3 Electron density maps of the key residues of the MYB-bHLH complexes.

a-f, 2mFo-DFc electron density maps of the residues important for the complex formation of WER-EGL3 (a,b), CPC-EGL3 (c,d) and MYB29-MYC3 (e,f). The electron density maps are contoured at 1.5, 1.5, and 1.0 sigma levels in WER-EGL3, CPC-EGL3 and MYB29-MYC3, respectively.

Extended Data Fig. 4 Mapping the interaction domains of CPC and EGL3.

a, Mapping the interacting regions of CPC and EGL3 by Y2H assay. b, ITC experiments showing the binding affinities between EGL3 and full-length CPC or CPC R3 (CPC 30–94).

Source data

Extended Data Fig. 5 Sequence alignment of MYB transcriptional factors homologous to WER or CPC in Arabidopsis.

a, Sequence alignment of the R2R3-type MYBs homologous to WER. b, Sequence alignment of R3-type MYBs homologous to CPC. The secondary structures are indicated at the top of the sequence alignment. The residues involved in binding to EGL3 are indicated by black arrows.

Extended Data Fig. 6 SDS-PAGE analysis of SEC experiments.

a, The SEC fraction of WER-EGL3 + CPC in Fig. 3a, while the same SDS-PAGE gel is displayed with high contrast. b-f, The SEC fractions of CPC-EGL3 (b), WER-EGL3 (c), CPC (d), WER (e) and EGL3 (f) in Fig. 3a are analyzed by SDS-PAGE, together with their corresponding SEC profiles. The experiments were repeated independently twice with similar results.

Source data

Extended Data Fig. 7 CPC Met49 residue is important to compete with WER for binding to EGL3.

a, ITC experiments showing the binding affinities between EGL3 and CPC mutants. b, ITC experiments showing the binding affinities between EGL3 and WER mutants.

Source data

Extended Data Fig. 8 Western blotting analysis.

The in vivo protein expression levels for spilt luciferase analysis in Fig. 3b are verified by Western blotting, and the experiments were repeated independently twice with similar results.

Source data

Extended Data Fig. 9 The competitive capability of WER against CPC to form a complex with EGL3.

a, Split-luciferase assay to detect the competition of WER against CPC to interact with EGL3. b, Quantitative measurement of the competitive capability of WER against CPC to form a complex with EGL3 by microscale thermophoresis (MST) assay, and the data were presented as mean values ± SD of three independent experiments (n = 3).

Source data

Extended Data Fig. 10 Bio-Layer Interferometry (BLI) assay.

BLI experiments are performed to test the binding affinities of WER-EGL3 and CPC-EGL3.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–17, Tables 1–6 and Source data for Supplementary Fig. 1.

Reporting Summary.

Source data

Source Data Fig. 1

Raw data of ITC.

Source Data Fig. 3

Unprocessed SDS-gel.

Source Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 1

Unprocessed SDS-gel.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 4

Raw data of ITC.

Source Data Extended Data Fig. 6

Unprocessed SDS-gels.

Source Data Extended Data Fig. 7

Raw data of ITC.

Source Data Extended Data Fig. 8

Unprocessed western blots.

Source Data Extended Data Fig. 9

Statistical source data.

Source Data Extended Data Fig. 10

Statistical source data.

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Wang, B., Luo, Q., Li, Y. et al. Structural insights into partner selection for MYB and bHLH transcription factor complexes. Nat. Plants 8, 1108–1117 (2022). https://doi.org/10.1038/s41477-022-01223-w

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