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Structures and mechanisms of the Arabidopsis cytokinin transporter AZG1

Nature Plants volume 10, pages 180–191 (2024)Cite this article

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Abstract

Cytokinins are essential for plant growth and development, and their tissue distributions are regulated by transmembrane transport. Recent studies have revealed that members of the ‘Aza-Guanine Resistant’ (AZG) protein family from Arabidopsis thaliana can mediate cytokinin uptake in roots. Here we present 2.7 to 3.3 Å cryo-electron microscopy structures of Arabidopsis AZG1 in the apo state and in complex with its substrates trans-zeatin (tZ), 6-benzyleaminopurine (6-BAP) or kinetin. AZG1 forms a homodimer and each subunit shares a similar topology and domain arrangement with the proteins of the nucleobase/ascorbate transporter (NAT) family. These structures, along with functional analyses, reveal the molecular basis for cytokinin recognition. Comparison of the AZG1 structures determined in inward-facing conformations and predicted by AlphaFold2 in the occluded conformation allowed us to propose that AZG1 may carry cytokinins across the membrane through an elevator mechanism.

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Fig. 1: Overall structure of AZG1.
Fig. 2: The dimeric assembly of AZG1.
Fig. 3: The ligand recognition of AZG1.
Fig. 4: The H-bond network near the substrate-binding site.
Fig. 5: The proposed model of AZG1.

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Data availability

Structure coordinates and cryo-EM density maps have been deposited in the Protein Data Bank under accession numbers PDB 8IRL and EMD-35678 for AZG1apo/pH7.4; PDB 8IRM and EMD-35679 for AZG1adenine/pH5.5; PDB 8WO7 and EMD-37681 for AZG1 T440Y/apo/pH7.4; PDB 8IRO and EMD-35681 for AZG1tZ/pH7.4; PDB 8WMQ and EMD-37658 for AZG1 tZ/pH5.5; PDB 8IRN and EMD-35680 for AZG1BAP/pH7.4; and PDB 8IRP and EMD-35682 for AZG1kinetin/pH7.4. The AZG1 structure predicted by AlphaFold2 was downloaded from the AlphaFold Database (https://alphafold.ebi.ac.uk/entry/Q9SRK7). Other structure coordinates analysed in the paper can be downloaded from the Protein Data Bank under accession numbers PDB 5DA0 for Deinococcus geothermalis SLC26Dg; PDB 6KI1 for Synechocystis BicA; PDB 5I6C for Aspergillus nidulans UapA; and 5XLS for Escherichia coli UraA occluded state. Additional data supporting the findings in this study are provided in the Supplementary Information. Source data are provided with this paper. Uncropped images for Extended Data Figs. 2a and 3a,g,m are supplied in Source Data.

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Acknowledgements

Single-particle cryo-electron microscopy data were collected at the Center of Cryo-Electron Microscopy at Zhejiang University and the Cryo-Electron Microscopy Facility of Hubei University. We thank X. Zhang and S. Chang for support with facility access and data acquisition. This work was supported in part by the National Key Research and Development Program of China (2022YFA1303400 to S.J.Z. and S.Q., 2020YFA0908501 to J.G. and 2020YFA0908400 to S.W.), Zhejiang Provincial Natural Science Foundation (LR19C050002 to J.G.), the Distinguished Young Scholars of Hubei Province (2022CFA078 to S.W.) and Fundamental Research Funds for the Central Universities. J.G. was supported by MOE Frontier Science Center for Brain Science and Brain–Machine Integration, Zhejiang University.

Author information

Author notes

  1. These authors contributed equally: Lingyi Xu, Wei Jia, Xin Tao.

Authors and Affiliations

  1. Department of Biophysics and Department of Neurology of the Fourth Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China

    Lingyi Xu, Fan Ye, Yan Zhang & Jiangtao Guo

  2. Nanhu Brain-computer Interface Institute, Hangzhou, China

    Lingyi Xu, Fan Ye, Yan Zhang & Jiangtao Guo

  3. National Engineering Laboratory of Intelligent Food Technology and Equipment, Zhejiang Key Laboratory for Agro-Food Processing, College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, Zhejiang, China

    Wei Jia & Yu Zhang

  4. Calibra Lab at DIAN Diagnostics, Key Laboratory of Digital Technology in Medical Diagnostics of Zhejiang Provinces, Hangzhou, Zhejiang, China

    Wei Jia

  5. State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, Hubei, China

    Xin Tao & Shan Wu

  6. State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, China

    Zhong Jie Ding & Shao Jian Zheng

  7. International Institutes of Medicine, the Fourth Affiliated Hospital, Zhejiang University School of Medicine, Yiwu, Zhejiang, China

    Shuai Qiao & Nannan Su

  8. NHC and CAMS Key Laboratory of Medical Neurobiology, MOE Frontier Science Center for Brain Science and Brain-Machine Integration, School of Brain Science and Brain Medicine, Zhejiang University, Hangzhou, Zhejiang, China

    Jiangtao Guo

  9. Department of Cardiology, Key Laboratory of Cardiovascular Intervention and Regenerative Medicine of Zhejiang Province, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China

    Jiangtao Guo

  10. Cancer Center, Zhejiang University, Hangzhou, Zhejiang, China

    Jiangtao Guo

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

    Jiangtao Guo

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  1. Lingyi Xu
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  3. Xin Tao
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Contributions

J.G., S.W., Yu Zhang and L.X. conceived and supervised the project. L.X., X.T. and Yan Zhang performed sample preparation, data acquisition and structure determination. L.X. and W.J. performed the functional assay. J.G., Yu Zhang, S.W., L.X., F.Y., S.J.Z., S.Q., N.S. and Z.J.D. performed structure data analysis. All authors participated in the manuscript preparation.

Corresponding authors

Correspondence to Lingyi Xu, Yu Zhang, Shan Wu or Jiangtao Guo.

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The authors declare no competing interests.

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Nature Plants thanks Veronica Maurino, Alexander Sobolevsky, Changlin Tian 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 Sequence alignment of Arabidopsis thaliana AZG1/2 with AZG1 from other representative plants.

Sequences are aligned with ClustalW. Secondary structural elements of Arabidopsis thaliana AZG1 are indicated above the sequence alignment. Amino acid residues involved in cytokinin binding are indicated by triangles in blue. Sequences for the listed AZG1 homologs are available at NCBI with the following accession numbers. AtAZG1 (Arabidopsis thaliana): AEE74982, AtAZG2 (Arabidopsis thaliana): AED95923, NtAZG1 (Nicotiana tabacum): XP_016473942, OsAZG1 (Oryza sativa): XP_015637355, ZmAZG1 (Zea mays): NP_001146825, TaAZG1 (Triticum aestivum): XP_044448085, GmAZG1 (Glycine max): XP_003533668, SlAZG1 (Solanum lycopersicum): XP_004240877, HvAZG1 (Hordeum vulgare): XP_044955314.

Extended Data Fig. 2 LC-MS/MS analysis of AZG1.

a, AZG1 expression detected by whole-cell western blot using anti-Flag tag antibody. n = 3 independent experiments. b, Typical UHPLC-MS/MS chromatograms for the analysis of tZ. c, Typical UHPLC-MS/MS chromatograms for the analysis of tZ-d5. d, Typical UHPLC-MS/MS chromatograms for adenine standards and adenine extracted from WT and mutant AZG1 protein samples. e, Comparison of the peak area of adenine extracted from WT and mutant AZG1 protein samples. Data are mean ± s.d., n = 3 biologically independent samples. Statistical analysis was performed using two-tailed t-tests. P = 0.0000685 for D393F, P = 0.0001124 for T440Y, and P = 0.0000450 for D393W/T440W double mutant. Differences were considered statistically significant at P < 0.05.

Source data

Extended Data Fig. 3 Structure determination of AZG1apo/pH7.4, AZG1adenine/pH5.5, and AZG1T440Y/apo/pH7.4.

a, Size-exclusion chromatography of AZG1apo/pH7.4 on Superose 6 (GE Healthcare) and SDS-PAGE analysis of the final sample. n = 3 independent experiments. b, Representative cryo-EM micrograph and 2D classification images of AZG1apo/pH7.4. n = 3 independent experiments. c, Flowchart of image processing for AZG1apo/pH7.4. d, The density map of AZG1apo/pH7.4 colored by local resolution. e, Angular distribution plot of particles included in the final C2-symmetric 3D reconstruction of AZG1apo/pH7.4. f, The Gold standard Fourier Shell Correlation (FSC) curve of the final 3D reconstruction, and the FSC curve for cross-validation between the map and the model of AZG1apo/pH7.4. g-l, Equivalent panels to the ones described above, for the structure determination of AZG1adenine/pH5.5. For g and h, n = 3 independent experiments. m-r, Equivalent panels to the ones described above, for the structure determination of AZG1T440Y/apo/pH7.4. For m and n, n = 3 independent experiments.

Source data

Extended Data Fig. 4 Structure determination of AZG1tZ/pH7.4, AZG1tZ/pH5.5, AZG1BAP/pH7.4 and AZG1kinetin/pH7.4.

a, Representative cryo-EM micrograph and 2D classification images of AZG1tZ/pH7.4. n = 3 independent experiments. b, Flowchart of image processing for AZG1tZ/pH7.4. c, The density map of AZG1tZ/pH7.4 colored by local resolution. d, Angular distribution plot of particles included in the final C2-symmetric 3D reconstruction of AZG1tZ/pH7.4. e, The Gold standard Fourier Shell Correlation (FSC) curve of the final 3D reconstruction, and the FSC curve for cross-validation between the map and the model of AZG1tZ/pH7.4. f-j, Equivalent panels to the ones described above, for the structure determination of AZG1tZ/pH5.5. For f, n = 3 independent experiments. k-o, Equivalent panels to the ones described above, for the structure determination of AZG1BAP/pH7.4. For k, n = 3 independent experiments. p-t, Equivalent panels to the ones described above, for the structure determination of AZG1kinetin/pH7.4. For p, n = 3 independent experiments.

Extended Data Fig. 5 Sample maps of AZG1 structures.

a-g, Sample maps at 14 transmembrane helices (TMs) of (a) AZG1apo/pH7.4, (b) AZG1adenine/pH5.5, (c) AZG1T440Y/apo/pH7.4, (d) AZG1tZ/pH7.4, (e) AZG1tZ/pH5.5, (f) AZG1BAP/pH7.4, and (g) AZG1kinetin/pH7.4. The density is shown as a mask around each TM at the level of 0.02 in UCSF ChimeraX. h-n, Density around the substrate-binding site in (h) AZG1apo/pH7.4, (i) AZG1adenine/pH5.5, (j) AZG1T440Y/apo/pH7.4, (k) AZG1tZ/pH7.4, (l) AZG1tZ/pH5.5, (m) AZG1BAP/pH7.4, and (n) AZG1kinetin/pH7.4 at the contour level of around 6 σ in Coot. The dashed circle marks no density of substrate in the map of AZG1T440Y/apo/pH7.4.

Extended Data Fig. 6 Comparisons of AZG1 structures determined in different conditions.

a-f, Superimposition of AZG1tZ/pH7.4 with (a) AZG1apo/pH7.4, (b) AZG1adenine/pH5.5, (c) AZG1T440Y/apo/pH7.4, (d) AZG1tZ/pH5.5, (e) AZG1 BAP/pH7.4, and (f) AZG1kinetin/pH7.4 subunit A structures in side view and top view. g, Structures of TM1-7 (blue) and TM8-14 (wheat) of AZG1 shown together or individually in the same orientation. h, Structural superimposition of TM1-7 (blue) and TM8-14 (wheat) within AZG1 in different orientations.

Extended Data Fig. 7 Structure of AZG1.

a, Topology and domain arrangement of AZG1. b, Structure of one subunit of AZG1apo/pH7.4, with individual elements colored as in a, and zoomed-in side view (left) and top view (right) of ECD.

Extended Data Fig. 8 Structure comparison of AZG1tZ/pH7.4 and AZG1AF.

a, The intracellular-facing cavity shown in the cut-in side view in AZG1tZ/pH7.4 (left) and AZG1AF (right). b, The radii along the potential transport path for AZG1tZ/pH7.4 (pink) and AZG1AF (purple). c, Top 10 tZ molecules docked into AZG1AF. Side view of the superimposition of AZG1tZ/pH7.4 (pink) and AZG1AF (purple) when they are aligned at the transport domain. ECD, TM5, 6, and 12 are omitted for clarity. The zoomed-in view of the substrate binding site reveals that the binding configurations of tZ (purple) in AZG1AF are similar to that of tZ (blue) in the AZG1tZ/pH7.4 structure.

Extended Data Fig. 9 Structure comparison of AZG1tZ/pH7.4, UraA, and UapA.

a, Side view of the superimposition of AZG1tZ/pH7.4 (pink) and UraA (salmon) when they are aligned at the transport domain and the zoomed-in view of substrate binding site. b, Side view of the superimposition of AZG1tZ/pH7.4 (pink) and AZG2AF (blue) predicted by AlphaFold2 when they are aligned at the transport domain and the zoomed-in view of substrate binding site. Substrates and residues related to proton coordination are shown as sticks. Wat1 is shown as red spheres. Hydrogen bonds are represented by red dashed lines. c, Side view (left) and top view (right) of the superimposition of inward-facing AZG1tZ/pH7.4 (pink) and inward-facing UapA (transport domain in light green and scaffold domain in dark green) when they are aligned at the whole transporter. d, Side view of the superimposition of transport domain (left) and scaffold domain (right) of AZG1tZ/pH7.4 and UapA with the same alignment in c. e, Side view (left) and top view (right) of the superimposition of occluded UraA (salmon) and occluded AZG1AF (transport domain in light blue and scaffold domain in dark blue) when they are aligned at the whole transporter. f, Side view of the superimposition of transport domain (left) and scaffold domain (right) of UraA and AZG1AF with the same alignment in e. g, Side view (left) and top view (right) of the superimposition of occluded UraA and inward-facing UapA when they are aligned at the scaffold domain. Red-green arrows indicate the vertical translation and lateral rotation of the transport domain. The green dot indicates the C2 symmetry axis. h, Side view of the superimposition of the transport domain (left) and scaffold domain (right) of UraA and UapA when they are aligned at the transport domain and scaffold domain, respectively. i, Vertical translation of conserved residues (Tyr288 in UraA and Phe406 in UapA) from occluded UraA to inward-facing UapA. Substrate and conserved residues are shown as sticks. TM3 and TM10 are shown in orange. j, The substrate binding sites in AZG1tZ/pH7.4 (pink) and AZG1AF (purple) which are aligned at the transport domain. k, The substrate binding sites in UraA (salmon) and UapA (green) which are aligned at the transport domain. Substrates and residues related to substrate coordination are shown as sticks.

Extended Data Table 1 Data collection and refinement statistics
Full size table

Supplementary information

Supplementary Information

Supplementary Fig. 1 and Table 1.

Reporting Summary

Supplementary Video 1

Molecular dynamics simulations of dimeric AZG1tZ/7.4. The substrate tZ molecules dissociate from the two substrate binding pockets within 60 ns, thereby confirming an inward-facing conformation of the AZG1 structures.

Supplementary Video 2

The transport model of AZG1 based on the structural comparison of inward-facing AZG1tZ/7.4 and occluded AZG1AF predicted by AlphaFold2.

Source data

Source Data Figs. 1, 3 and 4, and Extended Data Fig. 2

Statistical source data for Figs. 1b, 3m and 4g, and Extended Data Fig. 2e. Unprocessed western blots and gels for Extended Data Figs. 2a and 3a,g,m.

Source Data Extended Data Figs. 2 and 3

Unprocessed western blots and gels for Extended Data Figs. 2a and 3a,g,m.

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Xu, L., Jia, W., Tao, X. et al. Structures and mechanisms of the Arabidopsis cytokinin transporter AZG1. Nat. Plants 10, 180–191 (2024). https://doi.org/10.1038/s41477-023-01590-y

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