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Structural basis for the activity regulation of Salt Overly Sensitive 1 in Arabidopsis salt tolerance

Nature Plants volume 9, pages 1915–1923 (2023)Cite this article

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Abstract

The plasma membrane Na+/H+ exchanger Salt Overly Sensitive 1 (SOS1) is crucial for plant salt tolerance. Unlike typical sodium/proton exchangers, SOS1 contains a large cytoplasmic domain (CPD) that regulates Na+/H+ exchange activity. However, the underlying modulation mechanism remains unclear. Here we report the structures of SOS1 from Arabidopsis thaliana in two conformations, primarily differing in CPD flexibility. The CPD comprises an interfacial domain, a cyclic nucleotide-binding domain-like domain (CNBD-like domain) and an autoinhibition domain. Through yeast cell-based Na+ tolerance test, we reveal the regulatory role of the interfacial domain and the activation role of the CNBD-like domain. The CPD forms a negatively charged cavity that is connected to the ion binding site. The transport of Na+ may be coupled with the conformational change of CPD. These findings provide structural and functional insight into SOS1 activity regulation.

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Fig. 1: Architecture of the Arabidopsis SOS1.
Fig. 2: The interaction between the TMD and the IFD.
Fig. 3: Assembly of the various domains within the CPD.
Fig. 4: Functional elucidation for the four individual domains of SOS1.
Fig. 5: The Na+/H+ transport path of SOS1.
Fig. 6: A proposed model for the activity regulation of SOS1.

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Data availability

The 3D cryo-EM density maps have been deposited to the Electron Microscopy Data Bank (EMDB) under the accession codes EMD-36077 (SOS1 with stable CPD, Class 1) and EMD-36076 (SOS1 with flexible CPD, Class 2). The atomic coordinates for the corresponding model have been deposited to the Protein Data Bank (PDB) under the accession codes 8JD9 (https://doi.org/10.2210/pdb8JD9/pdb) (SOS1 with stable CPD, Class 1) and 8JDA (https://doi.org/10.2210/pdb8JDA/pdb) (SOS1 with flexible CPD, Class 2). Source data are provided with this paper.

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Acknowledgements

The cryo-EM data for SOS1 were collected at Shuimu BioSciences. We thank Z. Li of the Proteinomics Facility at China Agricultural University and X. Meng of the Proteinomics Facility at Tsinghua University for protein MS analysis. This work was supported by the National Key R&D Program of China (2022YFA1303400), the National Natural Science Foundation of China (32171188 to G.Y.), the Young Elite Scientists Sponsorship Program of the China Association for Science and Technology (to G.Y.) and the Chinese Universities Scientific Fund (2020RC008, 2020TC177, 2021RC012, 2022RC017, 2022TC144, 2023RC012 to G.Y.).

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  1. These authors contributed equally: Yanming Zhang, Jiaqi Zhou, Xuping Ni, Qinrui Wang.

Authors and Affiliations

  1. State Key Laboratory of Plant Environmental Resilience, Frontiers Science Center for Molecular Design Breeding, College of Biological Sciences, China Agricultural University, Beijing, China

    Yanming Zhang, Jiaqi Zhou, Xuping Ni, Yutian Jia, Xia Xu, Haoyang Wu, Peng Fu, Yan Guo & Guanghui Yang

  2. DP Technology, Beijing, China

    Qinrui Wang & Han Wen

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Contributions

G.Y. initiated and supervised the project. Y.Z., J.Z., Y.J. and H. Wu prepared the samples. X.N. prepared and analysed the phenotype of the sos1 mutant plants. Y.Z., X.N. and J.Z. performed the yeast growth experiment to reveal Na+ transport. Y.J., X.X. and Y.Z. collected the EM data. P.F. and G.Y. analysed the EM data and calculated the EM map. P.F. and G.Y. built and refined the atomic model. Q.W. and H. Wen performed and analysed the molecular dynamics simulation. G.Y. and Y.G. designed and analysed the biochemical experiments. All authors discussed the results. G.Y. wrote the manuscript.

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Correspondence to Guanghui Yang.

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Extended data

Extended Data Fig. 1 Alleles with sos1 mutants are sensitive to salt stress.

a, Seven mutants reduce the salt tolerance of Arabidopsis7. The location of sos mutants in protein SOS1 and amino acid(s) change are listed. Asterisk represents the translation stop. The location of these mutants is individually labelled. b, Salt sensitivity analysis of gl1 (WT), sos1-1, sos1-3, sos1-6, sos1-8, sos1-9, sos1-11 and sos1-12. Six-day-old seedlings grown on half-strength Murashige and Skoog (1/2 MS) medium were transferred to 1/2 MS medium or 1/2 MS medium containing 50 mM NaCl. Photographs were taken after 6-day treatment. c, Primary root length and fresh weight analysis of seedlings in b. Error bars represent the mean ± SD, n = 15 (three seedlings as a group were weighed together, and the average of each group was used for analysis), two-sided Student’s t test, *p < 0.05, ****p < 0.0001. (Exact P values of primary root length are 0.5512 for gl1, 3E-15 for sos1-11, the others are <1E-15; Exact P values of fresh weight are 0.3318, 6.03E-13, 1.19E-11, 2.38E-11, 7.62E-13, 2.65E-13, 2.33E-8, 5.82E-9 correspondingly). d, Net Na+ flux analysis of gl1 (WT), sos1-1, sos1-3, sos1-6, sos1-8, sos1-9, sos1-11 and sos1-12 by NMT. Seven-day-old seedlings grown on 1/2 MS medium were transferred to 1/2 MS medium with 50 mM NaCl for 12-h treatment, then the net Na+ flux was tested at the meristem zone. The flux data were shown every 12 s over 6 min. Based on the methods mentioned above, the positive net Na+ fluxes indicate the net Na+ efflux. Error bars represent the mean ± SD, n = 8. e, Calculated net Na+ fluxes in c Error bars represent the mean ± SD, n = 8. Different letters represent significant differences at p < 0.01 (one-way ANOVA). Exact P values are listed in Source Data Extended Data Fig. 1.

Source data

Extended Data Fig. 2 Protein purification of Arabidopsis thaliana SOS1.

A representative gel-filtration chromatography of SOS1. The peak fractions were visualized on SDS–PAGE by Coomassie staining. kDa, kilodaltons. MW, molecular weight marker. We have purified the protein three times and got similar results.

Extended Data Fig. 3 Cryo-EM data processing and analysis for SOS1 2class1.

a, A flowchart of the EM data processing by CryoSPARC46. 9060 micrographs were selected after CTF estimation and motion correction from 9098 movie stacks. b, Representative results of 2D classification of Class1 after particle picking and extraction. The red arrow indicates the stable CPD of Class1. c, The overall resolution of the reconstruction for SOS1 class1 is estimated to be 2.87 Å. d, Angular distribution of the particles used for reconstruction of SOS1. e, Local resolution estimation in Å of the SOS1 class1. f, FSC curves of the refined model versus the overall 2.87 Å map that it was refined against (black); of the models refined against the first half maps versus the same maps (purple); and of the models refined against the first half maps versus the second half maps (green). The small difference between the red and green curves indicates that the refinement of the atomic coordinates did not suffer from overfitting.

Extended Data Fig. 4 Cryo-EM data processing and analysis for SOS1 class2.

a, A flowchart of the EM data processing by CryoSPARC46. Similar to class1, class2 particles were separated from the same whole 1,035,234 particles. b, Representative results of 2D classification of Class2 in which no stable CPD seen. The red arrow indicates the flexible CPD of Class2. c, The overall resolution of the reconstruction for SOS1 class2 is estimated to be 3.67 Å. d, Angular distribution of the particles used for reconstruction of SOS1. e, Local resolution estimation in Å of the SOS1 class1. Although the resolution is inferior to class1, it is enough to build the distinct TMD. f, FSC curves of the refined model versus the overall 3.67 Å map that it was refined against (black); of the models refined against the first half maps versus the same maps (purple); and of the models refined against the first half maps versus the second half maps (green). The small difference between the red and green curves indicates that the refinement of the atomic coordinates did not suffer from overfitting.

Extended Data Fig. 5 Representative cryo-EM density for SOS1.

a, The local EM density map for SOS1 Class1. b, The local EM density maps for SOS1 Class2. The contour level of the EM density is 6 σ. The mainchain and the side chain of representative residues are shown in sticks. The figure was prepared by UCSF ChimeraX52,53 and PyMOL30.

Extended Data Fig. 6 Comparison of Class1 and Class2.

a, Comparison of cryo-EM maps of SOS1 Class1 and Class2 colored with grey and pink respectively. In the low-pass filtered maps, the relative downward motion of its CPD can be observed in the average densities. The arrows indicated the moving directions. b, Structural alignment of the TMD of Class1 and Class2. Compared to Class1, the shift of TM13 in Class2 triggers a slight movement of the TMD away from the symmetry axis of the dimer.

Extended Data Fig. 7 Dimeric assembly of SOS1 and cryo-EM analysis of the SOS1Δ998.

a, Co-purified phospholipids are located between the dimer domains and stabilize the dimer through hydrophobic effects. The contour level of the lipid density is 5 σ. b-c, Views from extracellular or intracellular side of SOS1. d, Structure alignment of SOS1 TMD with human NHE1 and Equus caballus NHE919,20. Superimposition of the dimeric TMD of SOS1 and NHE1 or NHE9 reveal an RMSD of 2.691 Å (over 534 Cα atoms) or 3.526 Å (over 570 Cα atoms), respectively. e, The two IFDs intertwine with each other. The nine helices are labeled as α1-α9. f, The result of 2D classification and the 3D reconstruction reveals highly flexible CPD and TMD of SOS1Δ998.

Extended Data Fig. 8 The Na+ coordination and putative conserved binding site in AtSOS1.

a, All-atom molecular dynamics simulations of SOS1 in the presence of high K+ concentration and varied Na+ concentrations. The ensemble of Na+ ions are shown as orange spheres while K+ ions are presented in palecyan. In the presence of high K+ concentration, both K+ and Na+ ions could move into the large cavity lined with negatively charged residues from CPD, reflecting the electrostatic feature of the CPD. b, Sequence alignment of SOS1 and other Na+/H+ exchangers revealed a conserved aspartic acid across different species. The equivalent residue in SOS1 is Asp201. Yeast growth on the medium with 25 mM NaCl reveals that the mutation Asp201Asn abolished Na+/H+ exchange compared to the wild-type SOS1 in the presence a reported constitutively active version of SOS2 (SOS2T168D/Δ308)53.

Extended Data Fig. 9 Structural mapping of mutants on SOS1 reveals molecular mechanism of functional regulation.

a, The locations of seven mutants in the structure and potential structural changes of SOS1. b, Closed-up view of sos1-3, sos1-8, sos1-9 and sos1-12. c, Sequence alignment of CNBD (-like) domains across different species reveals the conservation of Gly784.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
Full size table

Supplementary information

Reporting Summary

Supplementary Video 1

Movie of the 3D variability analysis of Class 1, illustrating the conformational dynamics occurring in the TMD and CPD regions of Class 1. It appears that the movement of the CPD potentially influences the conformation of the TMD.

Supplementary Video 2

Movie of the 3D variability analysis of Class 2, highlighting the dynamic behaviour of the TMD in Class 2, showing relative rotations between the core domain and the dimer domain. These observed changes may be associated with the high flexibility exhibited by the CPD of Class 2.

Source data

Source Data Extended Data Fig. 1

Statistical source data for Extended Data Fig. 1c,e.

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Zhang, Y., Zhou, J., Ni, X. et al. Structural basis for the activity regulation of Salt Overly Sensitive 1 in Arabidopsis salt tolerance. Nat. Plants 9, 1915–1923 (2023). https://doi.org/10.1038/s41477-023-01550-6

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