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Structure and mechanism of a bacterial sodium-dependent dicarboxylate transporter

Abstract

In human cells, cytosolic citrate is a chief precursor for the synthesis of fatty acids, triacylglycerols, cholesterol and low-density lipoprotein. Cytosolic citrate further regulates the energy balance of the cell by activating the fatty-acid-synthesis pathway while downregulating both the glycolysis and fatty-acid β-oxidation pathways1,2,3,4. The rate of fatty-acid synthesis in liver and adipose cells, the two main tissue types for such synthesis, correlates directly with the concentration of citrate in the cytosol2,3,4,5, with the cytosolic citrate concentration partially depending on direct import across the plasma membrane through the Na+-dependent citrate transporter (NaCT)6,7. Mutations of the homologous fly gene (Indy; I’m not dead yet) result in reduced fat storage through calorie restriction8. More recently, Nact (also known as Slc13a5)-knockout mice have been found to have increased hepatic mitochondrial biogenesis, higher lipid oxidation and energy expenditure, and reduced lipogenesis, which taken together protect the mice from obesity and insulin resistance9. To understand the transport mechanism of NaCT and INDY proteins, here we report the 3.2 Å crystal structure of a bacterial INDY homologue. One citrate molecule and one sodium ion are bound per protein, and their binding sites are defined by conserved amino acid motifs, forming the structural basis for understanding the specificity of the transporter. Comparison of the structures of the two symmetrical halves of the transporter suggests conformational changes that propel substrate translocation.

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Figure 1: Functional characterization and structure determination of the Na+-dependent dicarboxylate transporter VcINDY from Vibrio cholerae.
Figure 2: Structure of the VcINDY protomer.
Figure 3: Na + ion-binding sites in VcINDY.
Figure 4: Substrate-binding site in VcINDY.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession code 4F35.

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Acknowledgements

We are grateful to M. Punta and B. Rost for bioinformatics analysis of membrane transporters, to J. Love and B. Kloss for assistance in cloning, and to the staff at beamlines X4, X25 and X29 of the National Synchrotron Light Source in the Brookhaven National Laboratory and at the 23ID at the Advanced Photon Source at the Argonne National Laboratory for assistance in X-ray diffraction experiments, and to J. Llodra for help with artwork. We thank B. K. Czyzewski, W. A. Hendrickson, N. K. Karpowich, F. Mancia and J. J. Marden for discussions and for participating in synchrotron trips. This work was financially supported by National Institutes of Health grants U54-GM075026, R01-DK073973, R01-GM093825 and R01-MH083840.

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R.M. and D.-N.W. designed the project. R.M. did all the experiments, with assistance from G.G.G. in diffraction data processing, phasing and structure refinement, and from Q.L. in phasing. R.M. and D.-N.W. wrote the manuscript.

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Correspondence to Da-Neng Wang.

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The authors declare no competing financial interests.

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Mancusso, R., Gregorio, G., Liu, Q. et al. Structure and mechanism of a bacterial sodium-dependent dicarboxylate transporter. Nature 491, 622–626 (2012). https://doi.org/10.1038/nature11542

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