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Structure of Chlamydomonas reinhardtii LciA guided the engineering of FNT family proteins to gain bicarbonate transport activity

Nature Plants volume 12, pages 231–240 (2026)Cite this article

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Abstract

Engineering functional CO2-concentrating mechanisms into C3 crops holds great potential for enhancing photosynthetic efficiency. Limited CO2-inducible A (LciA), a chloroplast envelope bicarbonate channel belonging to the formate/nitrite transporter (FNT) family, is a key algal CO2-concentrating mechanism component and has been considered as a prime candidate for introduction into C3 plants. However, its application has been hindered by an incomplete mechanistic understanding. Here we report the cryogenic electron microscopy structure of Chlamydomonas reinhardtii LciA. Combining structural analysis and growth assays, we determined key residues governing substrate access and permeation, and identified two substitutions (K136A/A114F) that enhance LciA activity. We found that bicarbonate selectivity is governed by electrostatic coordination mediated by Lys220 and steric constraint imposed by Ala117 and Val267 within the selectivity filter. Leveraging these insights, we successfully engineered the bacterial FNT family nitrite channel NirC through site-directed mutagenesis to gain bicarbonate transport activity, and we characterized the bicarbonate transport capacity of the Chlamydomonas nitrite channels NAR1.1/NAR1.5, which were amenable to further enhancement. Taken together, our study establishes LciA as a fundamental template for engineering and identifying FNT proteins with bicarbonate transport capability, thereby greatly expanding the molecular toolkit for synthetic biology approaches aimed at boosting photosynthetic efficiency in both algae and crops.

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Fig. 1: Function characterization and overall structure of LciA.
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Fig. 2: Channel entrance of LciA.
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Fig. 3: Permeation pathway of LciA.
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Fig. 4: Selectivity filter of LciA.
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Fig. 5: Engineering the bacterial nitrite channel NirC to gain HCO3− transport activity.
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Fig. 6: Engineering the C. reinhardtii nitrite transporters NAR1.1 and NAR1.5 to gain HCO3− transport activity.
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Data availability

The cryo-EM density map and atomic coordinates of LciA have been deposited in the Electron Microscopy Data Bank and the Protein Data Bank under the accession numbers EMD-64722 and 9V2A, respectively. Source data are provided with this paper.

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Acknowledgements

We thank L. Liu from the University of Liverpool for critical reading and discussion of this manuscript. We thank H. Zhao and A. Dong at the cryo-EM centre of Fudan University and M. Zhang at the Cryo-EM Facility at the CAS Center for Excellence in Molecular Plant Sciences for their technical assistance on cryo-EM data collection. This work was supported by grants from the National Natural Science Foundation of China (nos 32530054 and 32025020 to P.Z. and no. 32401000 to Z.Y.), the Chinese Academy of Sciences (nos 317GJHZ2022023GC and XDB0630100 to P.Z.), the Shanghai Science and Technology Commission (no. 23310710100 to P.Z.), the National Key R&D Program of China (no. 2023YFA0914600 to J.H.), the Postdoctoral Fellowship Program of the China Postdoctoral Science Foundation (no. GZC20232665 to Z.Y.) and the Shanghai ‘Super Postdoctoral’ Incentive Program (no. 2023647 to Z.Y.).

Author information

Author notes

  1. These authors contributed equally: Jiaxin Guo, Zhao Yang.

Authors and Affiliations

  1. Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University, Shanghai, China

    Jiaxin Guo, Feifan Liu, Fang Yu & Jirong Huang

  2. Key Laboratory of Plant Carbon Capture, Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China

    Jiaxin Guo, Zhao Yang, Xue Zhang, Feifan Liu, Miaolian Ma & Peng Zhang

  3. Key Laboratory of Saline-Alkali Vegetation Ecology Restoration, Ministry of Education, College of Life Sciences, Northeast Forestry University, Harbin, China

    Jirong Huang

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  1. Jiaxin Guo
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Contributions

Z.Y. and J.G. designed and performed the bulk of the experiments. J.G. and Z.Y. carried out the protein expression and purification and sample preparation. Z.Y. and X.Z. carried out the cryo-EM data collection and structure determination. J.G., Z.Y. and F.L. carried out the growth assay. M.M. and F.Y. contributed to the protein purification. Z.Y., P.Z. and J.H. wrote the manuscript with input from the other authors. P.Z. and Z.Y. conceived the project.

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Correspondence to Zhao Yang, Jirong Huang or Peng Zhang.

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Nature Plants thanks Alistair McCormick, Takashi Yamano 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 LciA and FNT homologs from bacteria and parasite.

The structural architecture of LciA is shown on the top. Truncation variants LciAN39 (Δ1-38) and LciAN73 (Δ1-72) were designed based on sequence alignment with bacterial FNT homologs to delete the putative chloroplast-targeting peptide. Scissor symbols indicate the sites of N-terminal truncations corresponding to LciAN39 and LciAN73, with critical residues highlighted with boxes. Chlamydomonas reinhardtii LciA (UniProt: Q75NZ3), Salmonella typhimurium NirC (UniProt: E8XEH9), Escherichia coli FocA (UniProt: P0AC23), Clostridium difficile HSC (UniProt: Q186B7), Plasmodium falciparum PfFNT (UniProt: O77389).

Source data

Extended Data Fig. 2 Expression detected in Escherichia coli.

a–f, Western blots of LciA truncations and mutants. All mutations were performed in the LciAN39 truncation). (a) LciA full-length (FL) and truncations, related to Fig. 1b. (b) LciA K136 and R231 mutants, related to Fig. 2c,d. (c, d) LciA mutants in the permeation pathway, related to Fig. 3e. (e, f) LciA mutants in the selectivity filter, related to Fig. 4b,c. g–i, Western blots of bacterial and parasite FNT homologs. (g) NirC mutants, related to Fig. 5b. (h, i) FocA, HSC and PfFNT mutants, related to Extended Data Fig. 8d. j–m, Western blots of Chlamydomonas NAR1.1 and NAR1.5, related to Fig. 6c,f. (j, l) The NAR1.1 and NAR1.5 chimeras and mutants exhibit low expression levels, and strong signals are only detectable when they are immunoblotted individually. (k, m) When probed on the same membrane alongside LciAN39, only weak bands for NAR1.1 and NAR1.5 variants were visible, even though LciAN39 signal has been overexposed. Ponceau staining controls are included to verify equal loading. Each experiment was independently repeated three times with similar results.

Extended Data Fig. 3 Protein purification and structure determination.

a, Gel filtration profile on Superose 6 column and Coomassie-blue-stained SDS-PAGE analysis of LciA purified in buffer containing 0.03% DDM and 0.003% CHS. b, Gel filtration profile on Superose 200 column and Coomassie-blue-stained SDS-PAGE analysis of LciA-nanodiscs. For (a) and (b), Each experiment was independently repeated three times with similar results. c, Flowchart for Cryo-EM data processing. d, Representative cryo-EM micrograph (left) and 2D class averages (right). e, Local resolution estimation. The labels on the right are in unit Å. f, The gold-standard Fourier shell correlation curve. g, Cryo-EM densities of LciA protomer at a contour level of 6 σ.

Extended Data Fig. 4 Structural comparison.

a, b, Density map (a) and electrostatic potential surface (b) of LciA pentamer. The intermembrane-space view (left) and cut-open view from the membrane plane (right) are shown. Each protomer is colored distinctly. The elongated lipid-like densities in the central tunnel of pentamer are colored yellow. c, Electrostatic potential surface of FNT homologs, shown in periplasmic or extracellular view. Structural models of Escherichia coli FocA (PDB: 3KCU), Salmonella typhimurium NirC (PDB: 4FC4), Clostridium difficile HSC (PDB: 3TDO) and Plasmodium falciparum PfFNT (PDB: 7E26) are shown. d, Structural comparison of a protomer between LciA and FNT homologs.

Extended Data Fig. 5 Functional characterization of functional enhanced LciA and engineered NirC mutants in the E. coli Δcan strain at pH 7.0.

Phenotypes were recorded from the same plate at 24 h (a) and 36 h (b), with growth strength evaluated by dilution gradient and colony intensity. LciAN39 displayed a time-dependent improvement from weak to moderate growth, whereas the LciA mutants (K136A, K136A/A114F) sustained moderate growth, and NirC (I45A/I191V) consistently demonstrated the strongest growth.

Extended Data Fig. 6 Permeation pathway of LciA.

a, Cut-open electrostatic potential surface viewed from the membrane plane shows the channel architecture, including the external vestibule, central chamber and internal vestibule divided by two constrictions. The zoomed-in view shows the residues constituting the constrictions. b, Functional analysis of the LciA mutations in the E. coli Δcan strain at pH 7.0. All mutations were performed in the LciAN39 truncation.

Extended Data Fig. 7 Functional characterization of LciA mutations and chimeric NAR1.1/NAR1.5 in the E. coli Δcan strain.

a, Functional characterization of the A117S/V267I mutant in the E. coli Δcan strain at pH 9.0. b, Functional characterization of chimeric NAR1.1/NAR1.5 in the E. coli Δcan strain at pH 7.0.

Extended Data Fig. 8 Engineering of bacterial and parasite FNT homologs.

a–c, Structural alignments of LciA and EcFocA (a), CdHSC (b) and PfFNT (c) show the difference of the selectivity filter. Residues constituting the selectivity filter are shown as sticks. d, Functional characterization of engineered FNT-homolog variants in the E. coli Δcan strain at pH 9.0. e, Functional characterization of StNirC-I42F mutation (equivalent to LciA-A114F) and the NirC-I42F/I45A/I191V triple mutations in the E. coli Δcan strain at pH 7.0.

Extended Data Fig. 9 Sequence alignment of Chlamydomonas NAR1 homologs.

The structural architecture of LciA is shown on the top. Scissor symbols indicate the sites of N-terminal truncations corresponding to LciAN39 and LciAN73, with critical residues highlighted with boxes. Cr: Chlamydomonas reinhardtii. LciA/NAR1.2 (UniProt: Q75NZ3), NAR1.1 (UniProt: Q9LE25), NAR1.3 (UniProt: Q6IYG1), NAR1.4 (UniProt: Q6IYG4), NAR1.5 (UniProt: Q6IYG3), NAR1.6 (UniProt: Q6IYG2).

Extended Data Fig. 10 Engineering of Chlamydomonas NAR1 homologs.

a, c, e, Structural alignments of LciA with AlphaFold2-predicted NAR1.3 (a), NAR1.4 (c) and NAR1.6 (e) show the difference of the selectivity filter. Residues constituting the selectivity filter are shown as sticks. b, d, f, Functional characterization of engineered NAR1.3 (b), NAR1.4 (d) and NAR1.6 (f) in the E. coli Δcan strain at pH 9.0.

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Guo, J., Yang, Z., Zhang, X. et al. Structure of Chlamydomonas reinhardtii LciA guided the engineering of FNT family proteins to gain bicarbonate transport activity. Nat. Plants 12, 231–240 (2026). https://doi.org/10.1038/s41477-025-02200-9

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  • DOI: https://doi.org/10.1038/s41477-025-02200-9

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