Abstract
Adenosine triphosphate (ATP) is the principal energy currency of all living cells1,2. Metabolically impaired obligate intracellular parasites, such as the human pathogens Chlamydia trachomatis and Rickettsia prowazekii, can acquire ATP from their host cells through a unique ATP/adenosine diphosphate (ADP) translocator, which mediates the import of ATP into and the export of ADP and phosphate out of the parasite cells, thus allowing the exploitation of the energy reserves of host cells (also known as energy parasitism). This type of ATP/ADP translocator also exists in the obligate intracellular endosymbionts of protists and the plastids of plants and algae and has been implicated to play an important role in endosymbiosis3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31. The plastid/parasite type of ATP/ADP translocator is phylogenetically and functionally distinct from the mitochondrial ATP/ADP translocator, and its structure and transport mechanism are still unknown. Here we report the cryo-electron microscopy structures of two plastid/parasite types of ATP/ADP translocators in the apo and substrate-bound states. The ATP/ADP-binding pocket is located at the interface between the N and C domains of the translocator, and a conserved asparagine residue within the pocket is critical for substrate specificity. The translocator operates through a rocker-switch alternating access mechanism involving the relative rotation of the two domains as rigid bodies. Our results provide critical insights for understanding ATP translocation across membranes in energy parasitism and endosymbiosis and offer a structural basis for developing drugs against obligate intracellular parasites.
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Fig. 1: Structure of AtNTT1.
Fig. 2: Substrate-binding site of AtNTT1.
Fig. 3: Structure of CpNTT1.
Fig. 4: Conformational changes of the ATP/ADP translocator.
Data availability
The accession number of the Protein Data Bank coordinate of the mitochondrial ATP/ADP translocator used for structural comparison is 1OKC. The cryo-EM density maps of AtNTT1 and CpNTT1 have been deposited in the Electron Microscopy Data Bank, and the accession numbers are EMD-61117, EMD-61118, EMD-61119, EMD-61121 and EMD-61120. The corresponding atomic coordinates have been deposited in the Protein Data Bank, and the accession numbers are 9J3J, 9J3L, 9J3M, 9J3O and 9J3N.
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Acknowledgements
We thank X. Zhang at the Center of Cryo-Electron Microscopy at Zhejiang University for help with cryo-EM data collection, the Cryo-EM Facility at Westlake University for providing support for cryo-EM data collection, the State Key Laboratory of Genetic Engineering Cryo-EM Facility of Fudan University for sample imaging and M. Zhang at the Cryo-EM Facility at the CAS Center for Excellence in Molecular Plant Sciences for help with sample imaging. This study was made possible by support from the CAS Center for Excellence in Molecular Plant Sciences, the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB0630101), the CAS Pioneer Hundred Talents Program and the Shanghai Basic Research Project of Science and Technology Innovation Action Plan (23JC1403900) to M.F. X.W. was supported by Westlake Education Foundation. J.Z. was supported by startup funding from Fudan University. N.S. was supported by Fundamental Research Funds for the Central Universities.
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Author notes
These authors contributed equally: Huajian Lin, Jian Huang, Tianming Li, Wenjuan Li
Authors and Affiliations
CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China
Huajian Lin, Tianming Li, Wenjuan Li, Yutong Wu, Tianjiao Yang, Yuwei Nian, Ruiying Wang, Xiaohui Zhao & Minrui Fan
Key Laboratory of Plant Carbon Capture, Chinese Academy of Sciences, Shanghai, China
Huajian Lin, Tianming Li, Wenjuan Li, Yutong Wu, Tianjiao Yang, Yuwei Nian, Ruiying Wang & Minrui Fan
Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China
Jian Huang & Xudong Wu
Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China
Jian Huang & Xudong Wu
Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, China
Jian Huang & Xudong Wu
University of Chinese Academy of Sciences, Beijing, China
Yutong Wu & Tianjiao Yang
State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Department of Biochemistry and Biophysics, School of Life Sciences, Fudan University, Shanghai, China
Xiang Lin & Jinru Zhang
The Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu, China
Jiangqin Wang & Nannan Su
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Huajian Lin
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Contributions
H.L., J.H., T.L. and W.L. performed biochemical and cryo-EM studies. J.H. screened, expressed and purified the nanobodies under the supervision of X.W. H.L., T.L. and W.L. performed functional studies. Y.W., T.Y., Y.N., R.W. and X.Z. contributed to biochemical and functional studies. X.L., J.W., J.Z. and N.S. collected cryo-EM data. M.F., X.W., J.Z. and N.S. analysed the results. M.F. and X.W. directed the studies. M.F. wrote the Article with input from all authors.
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Correspondence to Nannan Su, Jinru Zhang, Xudong Wu or Minrui Fan.
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Extended data figures and tables
Extended Data Fig. 1 Biochemical and functional characterization of Arabidopsis thaliana plastid ATP/ADP translocator 1 (AtNTT1) and Chlamydia pneumoniae ATP/ADP translocator (CpNTT1).
a, Representative gel filtration profile of AtNTT1 and SDS-PAGE analysis of the purified AtNTT1. Data in a are representative of five independent experiments with similar results. b, Time course of AtNTT1-mediated ATP/ADP exchange (mean ± SEM, n = 4 independent experiments). c, ATP/ADP exchange activities of AtNTT1 wild type (WT) and loss-of-function mutant E246K (the data are normalized to WT; mean ± SEM, n = 4 independent experiments). d, Phosphate dependence of AtNTT1-mediated ATP/ADP exchange (normalized to WT; mean ± SEM, n = 4 independent experiments). e, Nucleotide preference of AtNTT1 (normalized to WT; mean ± SEM, n = 4 independent experiments). f, Co-elution of AtNTT1, Nb1 and anti-nanobody Fab on gel filtration column and SDS-PAGE analysis of the purified AtNTT1-Nb1-Fab complex. Data in f are representative of two independent experiments with similar results. g, Co-elution of AtNTT1, Nb2 and anti-nanobody Fab in the presence of ATP on gel filtration column and SDS-PAGE analysis of the purified AtNTT1-Nb2-Fab complex. Data in g are representative of two independent experiments with similar results. h, Co-elution of AtNTT1, Nb2 and anti-nanobody Fab in the presence of ADP and phosphate on gel filtration column and SDS-PAGE analysis of the purified AtNTT1-Nb2-Fab complex. Data in h are representative of two independent experiments with similar results. i, Representative gel filtration profile of CpNTT1 and SDS-PAGE analysis of the purified CpNTT1. Data in i are representative of five independent experiments with similar results. j, Time course of CpNTT1-mediated ATP/ADP exchange (mean ± SEM, n = 3 independent experiments). k, ATP/ADP exchange activities of CpNTT1 WT and loss-of-function mutant E157K (normalized to WT; mean ± SEM, n = 4 independent experiments). l, Co-elution of CpNTT1, Nb3 and anti-nanobody Fab in the presence of ATP on gel filtration column and SDS-PAGE analysis of the purified CpNTT1-Nb3-Fab complex. Data in l are representative of three independent experiments with similar results. For gel source data, see Supplementary Fig. 4.
Extended Data Fig. 2 Cryo-EM data processing of Arabidopsis thaliana and Chlamydia pneumoniae ATP/ADP translocators.
a, Representative cryo-EM images of AtNTT1 and CpNTT1. b, Flowcharts of data processing of AtNTT1 and CpNTT1.
Extended Data Fig. 3 Quality of cryo-EM density maps of Arabidopsis thaliana and Chlamydia pneumoniae ATP/ADP translocators.
Local resolution of the final map (left), gold-standard Fourier shell correlation (FSC) curves of the final map (middle) and angular distribution of particles for the final reconstruction (right) are shown for apo AtNTT1 (a), ATP-bound AtNTT1 (b), ADP/Pi-bound AtNTT1 (c), apo CpNTT1 (d) and ATP-bound CpNTT1 (e).
Extended Data Fig. 4 Cryo-EM density maps of Arabidopsis thaliana and Chlamydia pneumoniae ATP/ADP translocators.
Cryo-EM density maps of apo AtNTT1 (a), ATP-bound AtNTT1 (b), ADP/Pi-bound AtNTT1 (c), apo CpNTT1 (d) and ATP-bound CpNTT1 (e) are shown.
Extended Data Fig. 5 Topology and structural analysis of Arabidopsis thaliana ATP/ADP translocator.
a, Topology of AtNTT1. b, Structural comparison of outward-facing AtNTT1 (left panel) and mitochondrial ATP/ADP translocator (PDB 1OKC, right panel). The plastid ATP/ADP translocator is composed 12 TMs, while the mitochondrial ATP/ADP translocator contains six TMs.
Extended Data Fig. 6 Functional and structural analysis of the substrate-binding site of Arabidopsis thaliana ATP/ADP translocator.
a, ATP uptake activities of AtNTT1 variants with mutations at the substrate-binding site (the data are normalized to WT; mean ± SEM, n = 3 independent experiments). b-c, ATP/ADP exchange activities (b) and ATP uptake activities (c) of AtNTT1 variants with mutations on negatively charged residues of the substrate-binding site (normalized to WT; mean ± SEM, n = 4 independent experiments in b and n = 3 independent experiments in c). d, Comparison of the binding poses of ADP in the AtNTT1-ADP/PiIPO structure and ATP in the AtNTT1-ATPIPO structure. ADP and ATP are shown as cyan and grey sticks, respectively. e, ATP/ADP exchange activities of AtNTT1 under various concentrations of ADP (mean ± SEM, n = 3 independent experiments). f, Km for ADP and Vmax of AtNTT1-WT and mutants. g, Melting curves of AtNTT1 in the presence of different nucleotides and anions (the data are normalized to 10 °C; mean ± SEM, n = 3 independent experiments). Tryptophan-based fluorescence-detection size-exclusion chromatography (Trp-based FSEC) was used to determine the melting temperature of AtNTT1 under different conditions. The concentration of the nucleotides used is 100 µM, and the concentration of the anions used is 5 mM. h, Increase of the melting temperature of AtNTT1 by various nucleotides and anions (mean ± SEM, n = 3 independent experiments). i, Phosphate can increase the affinity of ADP (the data are normalized to unheated; mean ± SEM, n = 3 independent experiments). The apparent Kd values of ATP, ADP, and ADP in the presence of 5 mM phosphate determined using the thermal shift assay are 13.3 µM, 11.9 µM and 1.3 µM, respectively.
Extended Data Fig. 7 Chemical structures of various nucleotides and substrate specificities of Arabidopsis thaliana ATP/ADP translocator NTT1 and other plant plastid ATP/ADP translocators.
a, Chemical structures of ATP, GTP, CTP and UTP. b, Time course of AtNTT1-N282A mediated ATP/Pi exchange (mean ± SEM, n = 3 independent experiments). c, ATP/Pi exchange activities of AtNTT1-N282A under various concentrations of phosphate (mean ± SEM, n = 3 independent experiments). d, ATP/ADP and ATP/Pi exchange activities of AtNTT1 variants with mutations on N282 (the data are normalized to WT; mean ± SEM, n = 4 independent experiments). e-h, Left panels: transport activities of Arabidopsis thaliana NTT2 (AtNTT2) WT and N279A mutant (e), Spinacia oleracea (spinach) NTT (SoNTT) WT and N281A mutant (f) as well as Solanum tuberosum (potato) NTT (StNTT1 and StNTT2) WT and mutant (N280A in StNTT1 and N274A in StNTT2) (g-h) towards different substrates (normalized to WT; mean ± SEM, n = 4 independent experiments). Right panels: their ATP/NTP exchange activities (mean ± SEM, n = 4 independent experiments).
Extended Data Fig. 8 Topology, structural and functional analysis of Chlamydia pneumoniae ATP/ADP translocator.
a, Topology of CpNTT1. b, Structural comparison of the N-domains and C-domains of CpNTT1 and AtNTT1. c-d, Comparison of the ATP-binding modes in CpNTT1-ATPIO and AtNTT1-ATPIPO structures. e-f, ATP/ADP exchange activities (e) and ATP uptake activities (f) of CpNTT1 variants with mutations at the substrate-binding site (the data are normalized to WT; mean ± SEM, n = 4 independent experiments in e and n = 3 independent experiments in f). g, ATP/ADP exchange activities of CpNTT1 under various concentrations of ADP (mean ± SEM, n = 3 independent experiments). h, Km for ADP and Vmax of CpNTT1-WT and the N193A mutant. i, Transport activities of CpNTT1 WT and N193A mutant towards different substrates (normalized to WT; mean ± SEM, n = 4 independent experiments). j, ATP/NTP exchange activities of CpNTT1 WT and N193A mutant (mean ± SEM, n = 4 independent experiments). k, Transport activities of CpNTT1 WT and N193G mutant towards different substrates (normalized to WT; mean ± SEM, n = 4 independent experiments). l, ATP/NTP exchange activities of CpNTT1 WT and N193G mutant (mean ± SEM, n = 4 independent experiments). m, ATP/Pi exchange activities of AtNTT1-N282A and CpNTT1-N193A at different time points (mean ± SEM, n = 3 independent experiments).
Extended Data Fig. 9 Structural features of Arabidopsis thaliana and Chlamydia pneumoniae ATP/ADP translocators and their conformational changes.
a, AtNTT1 surface viewed from the intermembrane space side. The positively charged outward-facing cavity is indicated by an orange dashed circle. The surface is coloured by electrostatic potential (red, −5 kT e−1; blue, +5 kT e−1). b, Structure of CpNTT1OO viewed from the periplasmic side (left panel) and the cytoplasmic side (right panel). The positively charged residues at the inner sides of N-domain (palegreen) and C-domain (lightblue) are shown as sticks. c, Structure of AtNTT1-ATPIPO viewed from the stromal side (left panel) and the intermembrane space side (right panel). d, Structure of AtNTT1-ADP/PiIPO viewed from the stromal side (left panel) and the intermembrane space side (right panel). e-f, The positions of salt-bridge residues D49 and K309 in the CpNTT1OO (e) and CpNTT1-ATPIO (f) structures. g, ATP/ADP exchange activities of CpNTT1 WT and salt-bridge mutants (the data are normalized to WT; mean ± SEM, n = 4 independent experiments). h, Structural superposition of AtNTT1-ATPOO and AtNTT1-ATPIPO based on their N-domains. i, Structural superposition of AtNTT1-ATPOO and AtNTT1-ATPIPO based on their C-domains. j, Hypothetical model of NTT1-mediated ATP/ADP translocation. The ATP/ADP translocator operates through a rocker-switch mechanism, with two domains (N-domain and C-domain) rotating as rigid bodies to open and close outside and inside gates upon substrate binding. Major conformational states and key substrate-binding features of NTT1 are depicted. In AtNTT1, Y493 forms π-π interaction with the adenine moiety of ATP/ADP, and multiple positively charged residues from the inner sides of N-domain and C-domain interact with the phosphate tail of ATP/ADP; in addition, N282 forms a hydrogen bond with the adenine and is important for substrate specificity. The substrate-bound outward-facing and occluded conformations are yet to be captured experimentally.
Extended Data Table 1 Cryo-EM data collection, refinement, and validation statistics
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Supplementary information
Supplementary Figures
Supplementary Figs. 1–4.
Reporting Summary
Supplementary Video 1
Conformational changes of the plastid/parasite type of ATP/ADP translocator during transport. This video showcases the morphing of the structures of apo CpNTT1 and ATP-bound CpNTT1. The morph illustrates the conformational transition between the outward-facing state, where the central cavity is open to the periplasm; the occluded state, where the central substrate-binding site is inaccessible from either side of the membrane; and the inward-facing state, with the cavity open to the cytoplasmic side.
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Lin, H., Huang, J., Li, T. et al. Structure and mechanism of the plastid/parasite ATP/ADP translocator. Nature (2025). https://doi.org/10.1038/s41586-025-08743-3
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Received:05 August 2024
Accepted:05 February 2025
Published:12 March 2025
DOI:https://doi.org/10.1038/s41586-025-08743-3
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