Structure and activation mechanism of the rice salt overly sensitive 1 (sos1) na+/h+ antiporter

ABSTRACT Salinity is one of the most severe abiotic stresses that adversely affect plant growth and agricultural productivity. The plant Na+/H+ antiporter Salt Overly Sensitive 1 (SOS1)

located in the plasma membrane extrudes excess Na+ out of cells in response to salt stress and confers salt tolerance. However, the molecular mechanism underlying SOS1 activation remains

largely elusive. Here we elucidate two cryo-electron microscopy structures of rice (_Oryza sativa_) SOS1, a full-length protein in an auto-inhibited state and a truncated version in an

active state. The SOS1 forms a dimeric architecture, with an NhaA-folded transmembrane domain portion in the membrane and an elongated cytosolic portion of multiple regulatory domains in the

cytoplasm. The structural comparison shows that SOS1 adopts an elevator transport mechanism accompanied by a conformational transition of the highly conserved Pro148 in the unwound

transmembrane helix 5 (TM5), switching from an occluded conformation in the auto-inhibited state to a conducting conformation in the active state. These findings allow us to propose an

inhibition–release mechanism for SOS1 activation and elucidate how SOS1 controls Na+ homeostasis in response to salt stress. Access through your institution Buy or subscribe This is a

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OF THE PLASMA MEMBRANE NA+/H+ ANTIPORTER SOS1 IN _ARABIDOPSIS_ Article Open access 26 July 2023 STRUCTURAL BASIS FOR THE ACTIVITY REGULATION OF SALT OVERLY SENSITIVE 1 IN _ARABIDOPSIS_ SALT

TOLERANCE Article 26 October 2023 STRUCTURAL INSIGHTS INTO ION SELECTIVITY AND TRANSPORT MECHANISMS OF _ORYZA SATIVA_ HKT2;1 AND HKT2;2/1 TRANSPORTERS Article 03 April 2024 DATA

AVAILABILITY All data generated or analysed in this paper are presented in the main text, figures, extended data figures and supplementary videos or are available from the corresponding

author upon request. The cryo-EM maps of the _Os_SOS1 full-length (_Os_SOS1FL) and truncated versions (_Os_SOS1976) have been deposited in the Electron Microscopy Data Bank with accession

codes EMD-35775 and EMD-35950, respectively, and their structural models have been deposited in the PDB with accession codes 8IWO and 8J2M, respectively (Extended Data Table 1). Source data

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This project is financially supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA24020305 to Y.C.) and the National Key Research and Development

Program of China (2020YFA0509903 to Y.C. and 2021YFA1300702 to M.S.). AUTHOR INFORMATION Author notes * These authors contributed equally: Xiang-yun Zhang, Ling-hui Tang, Jia-wei Nie,

Chun-rui Zhang, Xiaonan Han. AUTHORS AND AFFILIATIONS * State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,

Beijing, China Xiang-yun Zhang, Ling-hui Tang, Jia-wei Nie, Chun-rui Zhang, Qi-yu Li, Li Qin, Mei-hua Wang, Xiahe Huang, Min Su, Yingchun Wang & Yu-hang Chen * University of Chinese

Academy of Sciences, Beijing, China Xiang-yun Zhang, Ling-hui Tang, Jia-wei Nie, Chun-rui Zhang, Qi-yu Li, Li Qin, Mei-hua Wang, Yingchun Wang, Rui-ming Xu, Qi Xie & Yu-hang Chen *

National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Xiaonan Han & Rui-ming Xu *

College of Grassland Science and Technology, China Agricultural University, Beijing, China Feifei Yu * State Key Laboratory of Plant Environmental Resilience, College of Biological

Sciences, China Agricultural University, Beijing, China Yan Guo * State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, The Innovative Academy of Seed

Design, Chinese Academy of Sciences, Beijing, China Qi Xie * National Center of Technology Innovation for Maize, State Key Laboratory of Crop Germplasm Innovation and Molecular Breeding,

Syngenta Group China, Beijing, China Qi Xie Authors * Xiang-yun Zhang View author publications You can also search for this author inPubMed Google Scholar * Ling-hui Tang View author

publications You can also search for this author inPubMed Google Scholar * Jia-wei Nie View author publications You can also search for this author inPubMed Google Scholar * Chun-rui Zhang

View author publications You can also search for this author inPubMed Google Scholar * Xiaonan Han View author publications You can also search for this author inPubMed Google Scholar *

Qi-yu Li View author publications You can also search for this author inPubMed Google Scholar * Li Qin View author publications You can also search for this author inPubMed Google Scholar *

Mei-hua Wang View author publications You can also search for this author inPubMed Google Scholar * Xiahe Huang View author publications You can also search for this author inPubMed Google

Scholar * Feifei Yu View author publications You can also search for this author inPubMed Google Scholar * Min Su View author publications You can also search for this author inPubMed Google

Scholar * Yingchun Wang View author publications You can also search for this author inPubMed Google Scholar * Rui-ming Xu View author publications You can also search for this author

inPubMed Google Scholar * Yan Guo View author publications You can also search for this author inPubMed Google Scholar * Qi Xie View author publications You can also search for this author

inPubMed Google Scholar * Yu-hang Chen View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS X.Z. performed protein purification, cryo-EM data

collection, yeast experiments and data analysis; L.T. and X. Han performed cryo-EM data collection, structural determination and structural analysis; C.Z. performed model building,

structural analysis and sequence analysis; J.N., X. Han, Q.L., X. Huang, L.Q. and M.W. performed experiments; F.Y., M.S., R.X., Y.W., Y.G. and Q.X. analysed data; Y.C. initiated the project,

planned and analysed experiments, supervised the research and wrote the manuscript with input from all authors. CORRESPONDING AUTHOR Correspondence to Yu-hang Chen. ETHICS DECLARATIONS

COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Plants_ thanks Jose M. Pardo, Huazhong Shi and the other, anonymous, reviewer(s)

for their contribution to the peer review of this work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and

institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 STRUCTURE-BASED SEQUENCE ALIGNMENTS FOR THE REPRESENTATIVE SOS1S FROM BOTH DICOTS AND MONOCOTS. The cryo-EM structure of OsSOS1

is used to restrict sequence gaps to inter-helical segments, with the superior coils and arrows defining extents of the secondary elements. The sequences include representative members from

dicots and monocots: Oryza sativa Japonica Group (XP_015619351.1), Zea mays (XP_008645741.2), Sorghum bicolor (XP_002443674.1), Aegilops tauschii (XP_020190641.1), Brachypodium distachyon

(XP_003576505.1), Dendrobium officinale (XP_020699162.1), Asparagus officinalis (XP_020256864.1), Musa acuminata subsp. malaccensis (XP_018686090.1), Phoenix dactylifera (XP_008798100.1),

Amborella trichopoda (XP_006849492.1), Arabidopsis thaliana (NP_178307.2), Brassica rapa (XP_009114217.1), Eutrema salsugineum (XP_006395824.1), Beta vulgaris subsp. vulgaris

(XP_010680103.2), Glycine max (NP_001244939.1), Daucus carota (XP_017226136.1), Cucumis melo (XP_008466844.1), Vitis vinifera (NP_001268140.1), Juglans regia (XP_018828660.1), Eucalyptus

grandis (XP_010066529.2). The highly conserved inhibitory motifs (motif-1 and motif-2) are shown in boxes. Some critical residues are highlighted as following: 1. The putative

phosphorylation residues are highlighted in cyan (S1113 and S1135 in OsSOS1). 2. The mutated residues are highlighted in yellow. EXTENDED DATA FIG. 2 STRUCTURAL DETERMINATION OF THE

_OS_SOS1FL. (A) The workflow for image processing of the _Os_SOS1FL. 2D classification of the _Os_SOS1FL particles from the cryo-EM sample are shown. (B) Fourier shell correlation (FSC)

curve suggests an overall resolution at 3.1 Å, as estimated using the 0.143 cut-off criterion (Left). The FSC curves for cross-validation between the model and the unmasked (Black) or masked

(Pink) cryo-EM maps of _Os_SOS1 (Right). (C) Representative cryo-EM density map for the TM segments of TMD, helical elements of HD, CNBD, CTD and auto-inhibitory motif segments. EXTENDED

DATA FIG. 3 STRUCTURAL DETERMINATION OF THE _OS_SOS1976. (A) The workflow for image processing of the _Os_SOS1976. 2D classification ns of the _Os_SOS1976 particles from the cryo-EM sample

are shown. (B) Fourier shell correlation (FSC) curve suggests an overall resolution at 3.4 Å, as estimated using the 0.143 cut-off criterion (Left). The FSC curves for cross-validation

between the model and the unmasked (Black) or masked (Pink) cryo-EM maps of _Os_SOS1976 (Right). (C) Representative cryo-EM density map for the TM segments of the TMD. EXTENDED DATA FIG. 4

DOMAIN ORGANIZATION OF _OS_SOS1 AND SALT TOLERANCE ASSAY. (A) Ribbons drawing of the _Os_SOS1 protomer is shown, orientated as in Fig. 1a. (B) Salt tolerance assay of the _Os_SOS1 and its

C-terminal truncated mutants in the Na+-extrusion defective yeast cells (_S. cerevisiae_ AXT3K). EXTENDED DATA FIG. 5 STRUCTURAL ANALYSIS OF THE TMD OF THE _OS_SOS1. (A) Superimposition of

the TMD (_Os_SOS1, as colored in Fig. 2a) with archaeal Na+/H+ exchanger NhaP from _Pyrococcus abyssii_ (pink), with an r.m.s.d. of 2.6 Å/386 superimposed Ca and 20% sequence identity. (B)

Superimposition of the TMD (_Os_SOS1, as colored in Fig. 2a) with human Na+/H+ exchanger _Hs_NHE1 from (light purple), with an r. m. s. d. of 2.9 Å/389 superimposed Ca and 26% sequence

identity. (C) Cavities within the TMD. Zoom view are shown, as indicated. Two endogenously bound lipid molecules at the dimeric interface are shown as sticks. (D) A conserved inter-helices

salt bridge (R341 _vs_ E171) and a kink (~18°) at the middle of TM4 in the TMD. EXTENDED DATA FIG. 6 STRUCTURAL ANALYSIS OF THE HD OF THE _OS_SOS1. (A) Ribbons drawing and domain topology of

the _Os_SOS1 HD, with one protomer in green and the other in grey. (B) Electrostatic potential at helical domain surface. Electronegative and electropositive potential are colored in

degrees of red and blue saturation, respectively. One protomer is drawn as ribbon diagram, and colored in green. Highly conserved charged residues at the dimeric HD interface and

ion-conducting pathway are shown in stick. Two conserved glycine-mediate kinks, H6 (~35°) and H7 (~34°), are indicated. EXTENDED DATA FIG. 7 STRUCTURE-BASED SEQUENCE ALIGNMENT OF CNBDS. The

structures for both CNBDs (_Os_SOS1 and HCN) have been superimposed and used to restrict sequence gaps to inter-secondary structural elements. Superior coils and arrows define extents of the

secondary structural elements in _Os_SOS1 CNBD (top) and HCN CNBD (bottom). Two loss-of-function mutations in the b-roll structure of the CNBD, including G777D (_sos1-8_) and G784E

(_sos1-9_), led to impaired transport function in _Arabidopsis_ are highlighted in magenta. The residues critical for cAMP binding in the HCN CNBD are highlighted in cyan. EXTENDED DATA FIG.

8 STRUCTURAL ANALYSIS OF THE DOMAIN INTERFACES. (A) The TMD-HD domain interface. The dimeric TMD is shown in ribbon drawing, with one protomer in light blue and the other one in grey. The

dimeric HD is shown as surface, colored with electrostatic potential. Electronegative and electropositive potential are shown in degrees of red and blue saturation, respectively. (B) Domain

organization of TMD (light bule) and HD (salmon), with a dihedral angle of ~26°. (C) The HD-CNBD domain interface. The dimeric HD is shown in ribbon drawing, with one protomer in green and

the other one in grey. The CNBD-CTD is shown as surface, colored with electrostatic potential. Electronegative and electropositive potential are shown in degrees of red and blue saturation,

respectively. EXTENDED DATA FIG. 9 COMPARISON OF THE TRUNCATED _OS_SOS1976 WITH THE AUTO-INHIBITED _OS_SOS1FL. (A, B) The elution profile of the truncated SOS1976 (magenta) and the

full-length _Os_SOS1FL (sky-blue) on size-exclusion column Superose 6 (Increase 10/300 GL). The truncated SOS1976 elutes at 13.8 ml, whereas the full-length _Os_SOS1FL elutes at 14.3 ml. The

high-molecular weight aggregates elute at avoid volume are indicated with *. The eluted fraction were analyzed by SDS-PAGE. (C) The superimposition of the auto-inhibited _Os_SOS1FL

structure and the truncated SOS1976 structure, based on their dimeric TMD portions (with an r.m.s.d. of 2.38 Å/848 superimposed Ca), reveals distinctive structural differences occurred in

the cytoplasmic regions. The structure of the truncated SOS1976 is represented as a density map, with the TMD portion depicted in purple and the unmodeled cytoplasmic portion in gray. The

cytosolic portion of the _Os_SOS1FL is represented in cartoon format (light blue). This comparison highlights a substantial conformational change upon SOS1 activation. The masks of the

detergent boundary are illustrated in white. SUPPLEMENTARY INFORMATION REPORTING SUMMARY SUPPLEMENTARY VIDEO 1 Comparison of plant SOS1, SOS1FL (PDB ID: 8IWO, auto-inhibited state) versus

SOS1976(PDB ID: 8J2M, active). SUPPLEMENTARY VIDEO 2 Comparison of human _Hs_NHE1, occluded conformation (PDB ID: 7dsx) versus conducting conformation (PDB ID: 7dsv). SUPPLEMENTARY DATA 1

Validation report for the full-length SOS1FL (auto-inhibited state). SUPPLEMENTARY DATA 2 Validation report for the truncated SOS1976 (constitutively active state). SOURCE DATA SOURCE DATA

FIG. 1 Unprocessed size-exclusion profile and SDS–PAGE gels for ED-9a/b. RIGHTS AND PERMISSIONS Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to

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terms of such publishing agreement and applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Zhang, Xy., Tang, Lh., Nie, Jw. _et al._ Structure and activation

mechanism of the rice Salt Overly Sensitive 1 (SOS1) Na+/H+ antiporter. _Nat. Plants_ 9, 1924–1936 (2023). https://doi.org/10.1038/s41477-023-01551-5 Download citation * Received: 15 May

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