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Structure of Arabidopsis SOQ1 lumenal region unveils C-terminal domain essential for negative regulation of photoprotective qH

Nature Plants volume 8, pages 840–855 (2022)Cite this article

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Abstract

Non-photochemical quenching (NPQ) plays an important role for phototrophs in decreasing photo-oxidative damage. qH is a sustained form of NPQ and depends on the plastid lipocalin (LCNP). A thylakoid membrane-anchored protein SUPPRESSOR OF QUENCHING1 (SOQ1) prevents qH formation by inhibiting LCNP. SOQ1 suppresses qH with its lumen-located thioredoxin (Trx)-like and NHL domains. Here we report structural data, genetic modification and biochemical characterization of Arabidopsis SOQ1 lumenal domains. Our results show that the Trx-like and NHL domains are associated together, with the cysteine motif located at their interface. Residue E859, required for SOQ1 function, is pivotal for maintaining the Trx–NHL association. Importantly, the C-terminal region of SOQ1 forms an independent β-stranded domain that has structural homology to the N-terminal domain of bacterial disulfide bond protein D and is essential for negative regulation of qH. Furthermore, SOQ1 is susceptible to cleavage at the loops connecting the neighbouring lumenal domains both in vitro and in vivo, which could be a regulatory process for its suppression function of qH.

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Fig. 1: Structure of SOQ1NHL.
Fig. 2: Structures of SOQ1NHL-CTD and SOQ1NHL(E859K)-CTD.
Fig. 3: Structure of SOQ1Trx(mut)-NHL and the interactions between the Trx-like and NHL domains.
Fig. 4: E859 is pivotal for stabilizing the Trx–NHL interface and for the suppression function of SOQ1.
Fig. 5: CTD is required for SOQ1 suppression function.
Fig. 6: Comparison of SOQ1 with DsbD.
Fig. 7: Truncated forms of SOQ1 accumulate in the thylakoid lumen.
Fig. 8: Proposed working model of SOQ1 in suppressing qH.

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Data availability

Atomic coordinates and crystallographic structure factors have been deposited in the protein data bank under accession codes: 7DJJ (SOQ1NHL), 7DJM (SOQ1NHL-CTD), 7DJK (SOQ1Trx(mut)-NHL) and 7DJL (SOQ1NHL(E859K)-CTD). Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding authors upon request. Sequence data from this article can be found in the Arabidopsis Genome Initiative (TAIR; https://www.arabidopsis.org/index.jsp) under accession numbers At1g56500 (SOQ1) and At3g47860 (LCNP).

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Acknowledgements

We thank M. Brooks for providing the SOQ1 expression vector and for critical discussion together with A. Hertle and K. Niyogi. We thank W. Schröder for help with lumen preparation and A. Johansson for assistance with Arabidopsis transformants characterization. We thank Y. Gao for assistance in plasmid construction and protein expression. We thank F. Sun for advice in cryo-EM sample preparation, and X. C. Zhang and L. He for discussion. We are grateful to the staff at the Shanghai Synchrotron Radiation Facility (Shanghai, China) for technical support during diffraction data collection. We thank Y. Wu from the Institute of Microbiology State Key Laboratory of Plant Genomics, CAS and Y. Chen from IBP for the MST experiment; L. Niu, X. Ding and M. Zhang from IBP, CAS for mass spectrometry. Cryo-EM work was performed at the Center for Biological Imaging (CBI, http://cbi.ibp.ac.cn), IBP, CAS. We thank B. Zhu and L. H. Cheng from CBI for their help with the cryo-EM data collection. The project was funded by the National Key R&D Program of China (2016YFA0502900 and 2017YFA0503702), the Strategic Priority Research Program of CAS (XDB27020106) and National Natural Science Foundation of China (31600609 and 31770778). X.P. is sponsored by the Youth Innovation Promotion Association at the Chinese Academy of Sciences (2018128). A.M. was supported by European Commission Marie Skłodowska-Curie Actions Individual Fellowship Reintegration Panel (845687). This research (J.H. and A.M.) was supported by grants to UPSC from the Knut and Alice Wallenberg Foundation (2016.0341 and 2016.0352), the Swedish Governmental Agency for Innovation Systems (2016-00504), by a starting grant to A.M. from the Swedish Research Council Vetenskapsrådet (2018-04150) and by a consortium grant from the Swedish Foundation for Strategic Research (ARC19-0051).

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Author notes

  1. Xiaowei Pan

    Present address: College of Life Science, Capital Normal University, Beijing, China

  2. These authors contributed equally: Guimei Yu, Jingfang Hao, Xiaowei Pan.

Authors and Affiliations

  1. National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, P.R. China

    Guimei Yu, Xiaowei Pan, Lifang Shi, Yong Zhang, Hongcheng Fan, Yang Xiao, Jizhong Lou, Wenrui Chang & Mei Li

  2. University of Chinese Academy of Sciences, Beijing, P.R. China

    Guimei Yu, Hongcheng Fan, Yang Xiao, Fuquan Yang, Jizhong Lou & Wenrui Chang

  3. Umeå Plant Science Centre (UPSC), Department of Plant Physiology, Umeå University, Umeå, Sweden

    Jingfang Hao & Alizée Malnoë

  4. Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing, P.R. China

    Jifeng Wang & Fuquan Yang

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Contributions

X.P., W.C., A.M. and M.L. designed the research. G.Y., X.P. and J.H. performed the research with assistance from L.S. and Y.X. Y.Z. and J.L. performed the MD simulations, J.W. and F.Y. analysed the cross-linking data and H.F. and G.Y. performed the cryo-EM experiment. All of the authors analysed and discussed the data, and G.Y., J.H., X.P., A.M. and M.L. wrote the paper, with input from all authors.

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Correspondence to Alizée Malnoë or Mei Li.

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Nature Plants thanks Patrice Hamel 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 Constructs of SOQ1 used in the present study.

(A) Domain structure of SOQ1. SOQ1 contains chloroplast transit peptide (cTP; green), HAD domain (yellow), transmembrane region (TM; blue), Trx-like domain (cyan) containing the CCINC motif, NHL domain (bright orange) and CTD (hot pink). Numbers indicate amino acid positions. (B) Constructs of SOQ1 truncations used for crystallization, crosslinking and cryo-EM. The region built in each crystal structure is shown below its corresponding construct (framed in one rectangular box). Mutated residues are indicated. (C) Construct of SOQ1 truncations used for plant transformant lines. (D) Constructs of SOQ1 truncations for microscale thermophoresis (MST) experiment. (E) Constructs of SOQ1 truncations used as controls for heterogeneous degradation.

Extended Data Fig. 2 The structures of NHL domain and CTD.

(A) The interaction between Blade 1 and the C-terminal tail of Blade 6 in NHL domain. The hydrogen bonds are formed between R590, G598, S606, S607 from N-ter of blade 1 (blue) and K900, Q903, P904,T906 from C-ter of blade 6 (red), and are shown by the black dotted lines. The residues involved in hydrogen bond interactions are shown in stick mode. (B) Structural superposition of NHL domain from SOQ1NHL (marine), SOQ1NHL-CTD (pink), SOQ1NHL(E859K)-CTD (cyan) and SOQ1Trx(mut)-NHL (yellow). The N-terminal helix (N-ter) as a part of TN-loop points upwards to link with the Trx-like domain in SOQ1Trx(mut)-NHL structure. (C) Structural superposition of CTD from SOQ1NHL-CTD (pink) and SOQ1NHL(E859K)-CTD (cyan). Two Cys residues C1006 and C1012 are shown as stick mode, and the distance (Å) between them is indicated.

Extended Data Fig. 3 Identification of the position of CTD in the SOQ1NHL-CTD structure.

(A) The seven symmetrically-related CTDs around one NHL domain in SOQ1NHL-CTD crystal. The NHL domain is shown in yellow. The CTD built in SOQ1NHL-CTD structure is shown in hot pink, and the other six symmetrically-related CTDs are shown in pink (the six symmetrically-related NHLs are omitted for clarity). The last traced residue (P907) in NHL domain and the first traced residue (T923) in CTDs are shown in stick-and-ball mode, and the distances (Å) between these two residues are indicated. (B) The scheme of BS3 crosslinked Lys pair. The BS3 is able to crosslink two Lys residues with the distance between their Cα atoms of approximately 24 Å. (C) The distances of cross-linked Lys pairs (K666/K678 from the NHL domain and K924/K927 from the seven CTDs).

Extended Data Fig. 4 Structural comparison of SOQ1Trx(mut)-NHL with reduced NHLRC2.

Superposition of SOQ1Trx(mut)-NHL (pink) and NHLRC2 (gray). Residue E859, R478, Y429, C431S and C434S in SOQ1Trx(mut)-NHL and the corresponding residues E533, R138, Y88, C90 and C93 in NHLRC2 are shown as sticks. The hydrogen bonds formed by E533 with Y88 and R138 are shown as black dashed lines and the distance (Å) is labeled. The distance (Å) between C431S and C434S is labeled. NHLRC2 shows two additional helices at the N-terminal region compared with SOQ1Trx(mut)-NHL. This corresponding fragment is present in SOQ1 sequence, but was not included in SOQ1Trx(mut)-NHL construct.

Extended Data Fig. 5 Structural model of SOQ1 lumenal domains formed by superposing SOQ1Trx(mut)-NHL and SOQ1NHL-CTD structures, aligned on their NHL domain.

The Trx-like domain, NHL domain and CTD are colored in cyan, bright orange and hot pink, respectively. The black dashed circle highlights the region where Trx-like domain overlaps with CTD. The four cross-linked Lys pairs with distances longer than 24 Å are indicated. The red arrow suggests the moving direction of the CTD region containing K952 and K957.

Extended Data Fig. 6 Cryo-EM analysis of SOQ1-LD(M).

(A) The representative cryo-EM micrographs of SOQ1-LD(M). Scale bar, 10 nm. (B) The representative of 2D class averages. (C) Plots showing the Euler angle distribution of the particles. (D) Different view of Cryo-EM reconstruction of SOQ1-LD(M). (E-F) Side (E) and top (F) view of the fitting of SOQ1Trx(mut)-NHL (cyan) and SOQ1NHL-CTD (magenta) crystal structures into the cryo-EM map. The red dotted circle in (D-F) indicates the density which may correspond to the CTD, but could not be fitted with the entire CTD, suggesting that CTD is mobile and partially occupies this area.

Extended Data Fig. 7 ΔCTD accumulation, localization and topology.

(A) ΔCTD localization in chloroplast sub-fractions (Thy: thylakoid, TM: thylakoid membrane after separating lumen, Lumen) in low light (LL) and after 6 h cold and high light treatment (cold HL). Samples were loaded at the same protein amount (3 µg proteins). Proteins were separated by SDS-PAGE and analyzed by immunodetection with antibodies against FLAG, PC, Lhcb4 or ATPb. ATPb is shown as loading control. Representative immunoblot from two independent biological experiments is shown. (B) ΔCTD localization and accumulation compared to wild type (Col-0) in low light (LL) and after 6 h cold and high light treatment (cold HL). Samples were loaded at the same protein amount (3 µg proteins). Proteins were separated by SDS-PAGE and analyzed by immunodetection with anti-SOQ1Trx. Star symbol (*) represents nonspecific band detected by the anti-Trx antibody, based on its absence in soq1-1 lumen samples. Representative immunoblot from three independent biological experiments is shown. (C) The topology of ΔCTD (3 µg chlorophyll) in ΔCTD T2-1, analyzed by immunodetection with anti-FLAG antibody. Protease protection assay on thylakoids treated with thermolysin (Th) in the presence or absence of Triton X-100 (Tr). The stroma-exposed PSI subunit D (PsaD) and the lumenal LCNP are shown as controls. Representative immunoblot from two independent biological experiments is shown. (D) Photosynthetic parameters Fo, Fm, Fv/Fm of Col-0, soq1-1 and three individual T2 ΔCTD lines at time 0 of the NPQ kinetics shown in Fig. 5c. Data represent means ± SD (n = 4 individuals).

Source data

Extended Data Fig. 8 The accumulation of SOQ1 and its truncated forms is similar in stress and non-stress conditions.

The relative quantities of SOQ1 in different fractionated thylakoid membrane preparations (A) and its truncated forms in the lumen (B). The quantities of SOQ1 in cold and high light (HL) were identified as a relative content to low light (LL). The sample was prepared using Yeda press (black dots) or sonication (blue dots) methods respectively. Thylakoid (Thy), membrane (Mb), and lumen samples (full-length and truncated forms) isolated from Col-0 were loaded at the same amount of total protein. Data represent means ± SD (n = 7 independent biological experiments (n = 6 for CTD quantification)).

Source data

Extended Data Table 1 Data collection and refinement statistics
Full size table
Extended Data Table 2 BS3 cross-linking results of SOQ1-LD and SOQ1-LD(E859K)
Full size table

Supplementary information

Supplementary Information

Supplementary Figs. 1–7 and additional Supplementary Fig. 1 (source data for Supplementary Fig. 2).

Reporting Summary

Supplementary Video 1

Molecular dynamics (MD) results of SOQ1Trx-NHL wild type.

Supplementary Video 2

Molecular dynamics (MD) results of SOQ1Trx-NHL(E859K).

Supplementary Data 1

Primer list for constructions and mutants.

Source data

Source Data Fig. 4c

Uncropped blots for Fig. 4c.

Source Data Fig. 5a,b

Uncropped blots for Fig. 5a,b.

Source Data Fig. 6F

Statistical source data for Fig. 6f: four times repeats of MST.

Source Data Fig. 7

Uncropped blots for Fig. 7.

Source Data Extended Data Fig. 7

Uncropped blots for Extended Data Fig. 7.

Source Data Extended Data Fig. 8

Statistical source data for Extended Data Fig. 8: seven repeats of truncated SOQ1 quantification.

Source Data Extended Data Table 2

Statistical source data for Extended Data Table 2: data of BS3 cross-linking results of SOQ1-LD and SOQ1-LD(E859K).

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Yu, G., Hao, J., Pan, X. et al. Structure of Arabidopsis SOQ1 lumenal region unveils C-terminal domain essential for negative regulation of photoprotective qH. Nat. Plants 8, 840–855 (2022). https://doi.org/10.1038/s41477-022-01177-z

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