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Structural insights into ion selectivity and transport mechanisms of Oryza sativa HKT2;1 and HKT2;2/1 transporters

Nature Plants volume 10, pages 633–644 (2024)Cite this article

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Abstract

Plant high-affinity K+ transporters (HKTs) play a pivotal role in maintaining the balance of Na+ and K+ ions in plants, thereby influencing plant growth under K+-depleted conditions and enhancing tolerance to salinity stress. Here we report the cryo-electron microscopy structures of Oryza sativa HKT2;1 and HKT2;2/1 at overall resolutions of 2.5 Å and 2.3 Å, respectively. Both transporters adopt a dimeric assembly, with each protomer enclosing an ion permeation pathway. Comparison between the selectivity filters of the two transporters reveals the critical roles of Ser88/Gly88 and Val243/Gly243 in determining ion selectivity. A constriction site along the ion permeation pathway is identified, consisting of Glu114, Asn273, Pro392, Pro393, Arg525, Lys517 and the carboxy-terminal Trp530 from the neighbouring protomer. The linker between domains II and III adopts a stable loop structure oriented towards the constriction site, potentially participating in the gating process. Electrophysiological recordings, yeast complementation assays and molecular dynamics simulations corroborate the functional importance of these structural features. Our findings provide crucial insights into the ion selectivity and transport mechanisms of plant HKTs, offering valuable structural templates for developing new salinity-tolerant cultivars and strategies to increase crop yields.

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Fig. 1: Cryo-EM determination of O. sativa HKT2;1 and HKT2;2/1 structures.
Fig. 2: Constriction sites in HKT2;1 and HKT2;2/1.
Fig. 3: Comparison between the SFs in HKT2;1 and HKT2;2/1.
Fig. 4: Electrophysiological studies on SF mutants of HKT2;2/1.
Fig. 5: Distinct permeation capabilities of Na+ and K+ ions in HKT2;1 and HKT2;2/1 revealed by MD simulations.
Fig. 6: A stable loop structure of the II–III linker in HKT2;1.
Fig. 7: A C-terminal tail crossing the dimeric interface.

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Data availability

The atomic coordinates and EM maps of HKT2;1 (PDB: 8K66; EMDB: EMD-36918) and HKT2;2/1 (PDB: 8K69; EMDB: EMD-36919) have been deposited in the Protein Data Bank (http://www.rcsb.org) and the Electron Microscopy Data Bank (https://www.ebi.ac.uk/pdbe/emdb/). Source data are provided with this paper.

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Acknowledgements

We thank the Cryo-EM Facility and Supercomputer Center of Westlake University for providing data collection and computation support. This work was supported by the National Natural Science Foundation of China (grant nos. 32122042 and 32071208 to H.S. and 32122040 and 31971040 to F.Y.), the Zhejiang Provincial Natural Science Foundation (grant no. LR20C050002 to F.Y.) and the Westlake Education Foundation (to H.S.).

Author information

Author notes

  1. These authors contributed equally: Xiaohui Wang, Xiaoshuai Shen, Yannan Qu, Heng Zhang, Chu Wang.

Authors and Affiliations

  1. Zhejiang Key Laboratory of Structural Biology, School of Life Sciences, Westlake University, Hangzhou, China

    Xiaohui Wang, Xiaoshuai Shen, Yannan Qu, Chu Wang & Huaizong Shen

  2. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China

    Xiaohui Wang, Xiaoshuai Shen, Yannan Qu, Chu Wang & Huaizong Shen

  3. Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, China

    Xiaohui Wang, Xiaoshuai Shen, Yannan Qu, Chu Wang & Huaizong Shen

  4. Department of Biophysics and Disease Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China

    Heng Zhang & Fan Yang

  5. Liangzhu Laboratory, Zhejiang University Medical Center, Hangzhou, China

    Heng Zhang & Fan Yang

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  1. Xiaohui Wang
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Contributions

H.S. conceived the project. X.W., X.S. and C.W. performed the molecular cloning, protein expression and purification, cryo-sample preparation and electron micrography data collection. Y.Q. conducted the structure reconstruction and model building. H.Z. carried out the electrophysiological experiments under the supervision of F.Y. All authors contributed to the data analysis. H.S. wrote the manuscript.

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Correspondence to Fan Yang or Huaizong Shen.

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Nature Plants thanks Ingo Dreyer, Maria Hrmova and Ming Zhou for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Sequence alignment of plant HKT and bacterial Ktr/Trk transporters.

The sequences of plant HKT and bacterial Ktr/Trk transporters were aligned with the Clustal Omega program82 and colored with ENDscript 283. Invariant residues are shaded in red, while conserved residues are colored red. Critical regions discussed in the paper are underlined and labeled. Red-filled circles indicate residues comprising the constriction site. Red-filled triangles indicate the conserved SF residues of ‘SGGG’ or ‘GGGG’. The secondary structures of rice HKT2;1 are depicted above aligned sequences. It is worth noting that the majority of Class I HKT transporters feature an SF sequence of ‘SGGG’ with the four residues originating from Domain I to IV, respectively. In contrast, most Class II HKT transporters possess an SF sequence of ‘GGGG’.

Extended Data Fig. 2 Protein purification, flowchart for EM data processing, and local-resolution maps.

(a) Size exclusion chromatography (SEC) profile of HKT2;2/1 and corresponding SDS-PAGE gel with coomassie-blue staining. Peak fractions of SEC were concentrated before preparing cryo-samples for data collection. (b) Flowchart for cryo-EM data processing. Please refer to the ‘Image processing’ section in the Methods for details. (c) The local-resolution maps for HKT2;1 and HKT2;2/1 were calculated by Relion and presented in Chimera.

Source data

Extended Data Fig. 3 EM densities for the transmembrane segments of HKT2;1 and HKT2;2/1.

The electron microscopy (EM) densities for the transmembrane segments of HKT2;1 (a) and HKT2;2/1 (b) are visualized using UCSF ChimeraX. Residues with large side chains are labeled. Critical residues discussed in the paper are highlighted with red shading.

Extended Data Fig. 4 EM densities for the SF, constriction sites, and II-III linker of HKT2;1 and HKT2;2/1.

(a) The electron microscopy (EM) densities corresponding to the selectivity filters (SF) are of high quality, which allows for accurate model building. Residues and the coordinated ions are labeled. Ser88 and Val243 in HKT2;1, and Gly88 and Gly243 in HKT2;2/1, which play crucial roles in ion selectivity, are highlighted in red shading. The densities are contoured at 12σ and 10σ for HKT2;1 and HKT2;2/1, respectively. (b) (c) The EM densities for constriction sites (b) and II-III linker of HKT2;1 (c) are contoured at 10σ and 8σ, respectively. All figures were prepared in PyMOL.

Extended Data Fig. 5 III-IV linker and bound lipids.

(a) The III-IV linker of HKT2;2/1, modeled with polyalanine, is visualized using ChimeraX. The linker and corresponding densities are colored red for clarity. Two perpendicular views are shown. (b) Both structures of HKT2;1 and HKT2;2/1 exhibit an abundance of bound lipids. HKT transporters are color-coded in the upper panel, while the bound lipids are visualized in light gray. In the lower panel, detailed densities for representative lipids are depicted. All figures are prepared in ChimerX.

Extended Data Fig. 6 Yeast complementation assay on residues at the constriction site or II-III linker in HKT2;2/1.

Yeast strains incapable of K+ absorption under low-K+ concentrations were transformed with plasmids containing wild-type (WT) HKT2;2/1 or its mutants carrying mutations of constriction site or II-III linker residues. The transformed yeasts were then subjected to growth tests under low-K+ concentrations. To ensure cross-validation, a series of dilutions of the seeding yeast were plated on two parallel rows. Two batches of yeast cultures, totaling four parallel rows, were plated on the same plate as biological replicates. E114Y mutation and substitution of the II-III linker with a flexible sequence consisting of 3 × GlyGlyGlySer (labeled as GS) in HKT2;2/1 substantially impaired the yeast’s growth on the K+-depleted medium compared with the wild type, while the effects of E114A, K517A, and R515A were either minimal or absent, suggesting that mutations of the constriction site residues to amino acids with bulky side chains may be necessary to effectively influence the ion conduction of the transporters. Experiments were repeated three times with representative results displayed.

Extended Data Fig. 7 Comparison of the selectivity filters from different channels.

The selectivity filters (SF) of KcsA, CNGA1, and NaK are illustrated, with the diagonal distances between the opposing coordinating oxygen atoms indicated.

Extended Data Fig. 8 Representative current traces of HKT2;1, HKT2;2/1 and their SF mutants.

Representative current traces of HKT2;1, HKT2;2/1, or their mutants carrying mutations of critical SF residues. The currents were elicited by a series of voltage steps from -120 mV to 60 mV with a 20-mV interval in bath solutions containing 135 mM NaCl or KCl.

Extended Data Fig. 9 Yeast complementation assay on SF mutants of HKT.

Yeast strains incapable of K+ absorption under low-K+ concentrations were transformed with plasmids containing wild-type (WT) HKT2;1 / HKT2;2/1 or their mutants carrying mutations of critical SF residues. The transformed yeasts were then subjected to growth tests under low-K+ concentrations. A series of dilutions of the seeding yeast were plated on two parallel rows for cross-validation. The results confirm the structural observations, highlighting the essential roles of Ser88 and Val243 in HKT2;1, and Gly88 and Gly243 in HKT2;2/1 in determining ion selectivity. Experiments were repeated three times with representative results displayed.

Extended Data Table 1 Statistics for data collection and structural refinement
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Supplementary Figs. 1 and 2.

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Source Data Fig. 4

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Source Data Extended Data Fig. 2

Unprocessed SDS–PAGE gel.

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Wang, X., Shen, X., Qu, Y. et al. Structural insights into ion selectivity and transport mechanisms of Oryza sativa HKT2;1 and HKT2;2/1 transporters. Nat. Plants 10, 633–644 (2024). https://doi.org/10.1038/s41477-024-01665-4

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