Structural mechanism of the active bicarbonate transporter from cyanobacteria

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Abstract

Bicarbonate transporters play essential roles in pH homeostasis in mammals and photosynthesis in aquatic photoautotrophs. A number of bicarbonate transporters have been characterized, among which is BicA—a low-affinity, high-flux SLC26-family bicarbonate transporter involved in cyanobacterial CO2-concentrating mechanisms (CCMs) that accumulate CO2 and improve photosynthetic carbon fixation. Here, we report the three-dimensional structure of BicA from Synechocystis sp. PCC6803. Crystal structures of the transmembrane domain (BicATM) and the cytoplasmic STAS domain (BicASTAS) of BicA were solved. BicATM was captured in an inward-facing HCO3-bound conformation and adopts a ‘7+7’ fold monomer. HCO3 binds to a cytoplasm-facing hydrophilic pocket within the membrane. BicASTAS is assembled as a compact homodimer structure and is required for the dimerization of BicA. The dimeric structure of BicA was further analysed using cryo-electron microscopy and physiological analysis of the full-length BicA, and may represent the physiological unit of SLC26-family transporters. Comparing the BicATM structure with the outward-facing transmembrane domain structures of other bicarbonate transporters suggests an elevator transport mechanism that is applicable to the SLC26/4 family of sodium-dependent bicarbonate transporters. This study advances our knowledge of the structures and functions of cyanobacterial bicarbonate transporters, and will inform strategies for bioengineering functional BicA in heterologous organisms to increase assimilation of CO2.

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Fig. 1: Functional characterization of BicA.
Fig. 2: The overall structure of BicATM.
Fig. 3: The substrate-binding site of BicA.
Fig. 4: Structural comparison of the substrate-binding sites.
Fig. 5: BicASTAS is required for the formation of BicA dimers.
Fig. 6: The structure and physiological importance of the BicA dimer.
Fig. 7: The transport mechanism of the Na+-dependent HCO3 transporters BicA and NBCe1.

Data availability

The atomic coordinates and structure factors of the transmembrane domain and STAS domain structures of BicA have been deposited in the Protein Data Bank with accession codes 6KI1 and 6KI2, respectively. The cryo-EM map of BicA has been deposited in the EMDB (EMD-9897).

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Acknowledgements

We thank the staff members of the BL19U1 and BL17U1 beamlines at the SSRF and the National Protein Science Center (Shanghai) for technical assistance in diffraction data and SEC–MALS (J. Zhang) data collection; the staff members at the cryo-EM center of University of Science and Technology of China and National Protein Science Center (Shanghai) for cryo-EM analysis and data collection; and the staff members at the core facility center of Institute of Plant Physiology and Ecology for X-ray diffraction analysis. We also thank the Liverpool Center for Cell imaging for technical assistance and provision of confocal microscopy. This work was supported by grants from the National Natural Science Foundation of China (grant no. 31861130356 to P.Z.), the Ministry of Science and Technology of China (grant no. 2015CB910900 to P.Z.), the Chinese Academy of Sciences (XDB27020103 and QYZDB-SSW-SMC006 to P.Z.); the Royal Society (NAF/R1/180433 to P.Z. and UF120411 and URF/R/180030 to L.-N.L.), the Biotechnology and Biological Sciences Research Council (BB/M024202/1 and BB/R003890/1 to L.-N.L.), an award from the University of Liverpool Technology Directorate Voucher Scheme (to F.H.) and the Leverhulme Trust Early Career Fellowship (ECF-2016-778 to F.H.).

Author information

P.Z. conceived the project. C.W. and P.Z. designed the experiments. C.W. performed the majority of the experiments; B.S. contributed to the LCP crystallization; X.Z. and X.H. performed the cryo-EM study; H.G. and F.Y. contributed to protein expression and crystallization; M.Z. contributed to transport assay analysis; X.C. and H.M. contributed to growth assay analysis; F.H., T.C. and L.-N.L. performed genetic GFP fusion and live-cell imaging. P.Z. and C.W. solved the crystal structures. P.Z., L.-N.L. and C.W. wrote the manuscript with inputs from all of the authors.

Correspondence to Peng Zhang.

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The authors declare no competing interests.

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Peer review information Nature Plants thanks Alistair McCormick and the other, anonymous, reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 BicA activity and purification.

a, Growth curves of Synechocystis sp. PCC6803 WT and Δ4/Δ5 mutants bubbled with air plus 1% CO2. b, H14CO3- uptake activity assayed using Synechocystis sp. PCC6803 WT and Δ4/Δ5 mutants. (Values are mean ± SD, n=3). c, SDS-PAGE shows the purification results of transmembrane domain truncations, and STAS domain truncations (d). The gels were stained with Coomassie blue. (Experiments were repeated at least three times).

Extended Data Fig. 2 Electron density and crystal packing of BicATM393.

a, 2Fo-Fc electron density of TM-1, -3, -8. The density is contoured at 1.5 σ. b, Crystal packing of BicATM. The crystal is of space group P2 and contains two molecules (colored in yellow and magenta) in the asymmetric unit. c, Fo-Fc electron densities of HCO3- and the metal ion (contoured at 2.0 σ). d, 2Fo-Fc electron density of monoolein (contoured at 1.0 σ).

Extended Data Fig. 3 Potential Na+ binding site.

a, Potential Na+-binding site in BicA. b, Na+-binding site in the Na+- dependent dicarboxylate transporter VcINDY (PDB 4F35). c, Na+- binding site in a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters LeuT (PDB 2A65).

Extended Data Fig. 4 The stable dimeric structure of BicASTAS.

a, Three-layered dimer of BicASTAS. The helices of the bottom layer and top layer are colored in dark blue, and the β-sheets of the middle layer are colored in cyan. b, SDS-PAGE shows the chemical cross- linking experimental results of different truncations of BicASTAS, Experiments were repeated at least three times. c/d, Topology of BicASTAS and SLC26DgSTAS. e, SLC26DgSTAS (colored in light blue) forms a heterodimer with the nanobody Nb5776 (colored in light grey) through forming a 10-stranded β-sheet (PDB 5IOF).

Extended Data Fig. 5 Oligomeric state of BicA full-length and transmembrane- domain.

Chemical cross-linking results of BicA (a) and transmembrane domain only (b). Three independent experiments have been carried with similar results.

Extended Data Fig. 6 Cryo-EM structure of BicA.

a, Representative micrograph. b, 2D class averages. c, Fourier shell correlation curve. d, Three different views of BicA reconstruction.

Extended Data Fig. 7 Sequence alignment of the STAS domain of Synechocystis sp. PCC6803 BicA and human SLC26A2-4.

The gene ID are 499176153 (Syn6803_BicA), 37590805 (Hp_SLC26A2), 19343676 (Hp_SLC26A3) and 119603820 (Hp_SLC26A4), respectively. The positions of human disease mutations in human SLC26A2-4 are indicated with blue triangles.

Extended Data Fig. 8 Structural comparison with other SLC family transporters.

a, Core domain superimposition of BicATM (orange) and NBCe1CTD (light grey). The RMSD is 2.5Å. b, Gate domain superimposition of BicATM (blue) and NBCe1CTD (light grey). The RMSD is 3.0Å. c, Superimposition of BicATM and NBCe1CTD. The RMSD is 3.8Å. d, Differences of the dimer interaction surface among AE1, NBCe1, Bor1, UapA and UraA transporters. The structure elements involved the dimerization are colored magenta in AE1, NBCe1 and Bor1 transporters (SLC4 family), and red in UapA and UraA transporters (SLC23 family).

Extended Data Fig. 9 PCR analysis of different BicA mutants.

Three independent experiments have been carried with similar results.

Supplementary information

Supplementary Table 1

Summary of the statistics for X-ray diffraction data collection, structure determination and refinement.

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Wang, C., Sun, B., Zhang, X. et al. Structural mechanism of the active bicarbonate transporter from cyanobacteria. Nat. Plants 5, 1184–1193 (2019) doi:10.1038/s41477-019-0538-1

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