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Structure of mitochondrial pyruvate carrier and its inhibition mechanism

Nature volume 641, pages 250–257 (2025)Cite this article

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Abstract

The mitochondrial pyruvate carrier (MPC) governs the entry of pyruvate—a central metabolite that bridges cytosolic glycolysis with mitochondrial oxidative phosphorylation—into the mitochondrial matrix1,2,3,4,5. It thus serves as a pivotal metabolic gatekeeper and has fundamental roles in cellular metabolism. Moreover, MPC is a key target for drugs aimed at managing diabetes, non-alcoholic steatohepatitis and neurodegenerative diseases4,5,6. However, despite MPC’s critical roles in both physiology and medicine, the molecular mechanisms underlying its transport function and how it is inhibited by drugs have remained largely unclear. Here our structural findings on human MPC define the architecture of this vital transporter, delineate its substrate-binding site and translocation pathway, and reveal its major conformational states. Furthermore, we explain the binding and inhibition mechanisms of MPC inhibitors. Our findings provide the molecular basis for understanding MPC’s function and pave the way for the development of more-effective therapeutic reagents that target MPC.

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Fig. 1: Overall structure of the human MPC1 and MPC2.
Fig. 2: MPC in complex with UK5099 or AKOS.
Fig. 3: Comparison of inhibitor-bound structures and the roles of the binding pocket residues.
Fig. 4: Inhibition of MPC by GW604714X.
Fig. 5: Pyruvate-bound structure and substrate-binding site.
Fig. 6: Conformational states and inhibition mechanism.

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Data availability

The MPC maps have been deposited in the Electron Microscopy Data Bank under EMD-48441, EMD-48442, EMD-48443, EMD-48444 and EMD-48445. The corresponding models have been deposited in the PDB under 9MNW, 9MNX, 9MNY, 9MNZ and 9MO0.

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Acknowledgements

We thank L. Montabana and M. Zaoralová at Stanford cEMc for help with electron microscopy data collection. Some of this work was performed at the Stanford-SLAC Cryo-EM Center (S2C2), which is supported by the US National Institutes of Health (NIH) Common Fund Transformative High-Resolution Cryo-Electron Microscopy programme (U24 GM129541). We thank the following S2C2 personnel for their invaluable support and assistance: Y. Liu. We also thank J. Jiang for technical advice; T. Chew for helpful discussions; and W. Frommer, L. Cheung and J. Rutter for sharing advice and/or reagents for initial setup of the yeast assay. This research is made possible by support from Stanford University, NIH R01GM117108 and NIH R35GM153424 (L.F.). E.J.F. is in the Sarafan ChEM-H Chemistry/Biology Interface Training Program and supported by NIH 5T32GM139791.

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Author notes

  1. These authors contributed equally: Zheng He, Jianxiu Zhang

Authors and Affiliations

  1. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA

    Zheng He, Jianxiu Zhang, Yan Xu, Eve J. Fine, Carl-Mikael Suomivuori, Ron O. Dror & Liang Feng

  2. Department of Bioengineering, Stanford University, Stanford, CA, USA

    Eve J. Fine

  3. Department of Computer Science, Stanford University, Stanford, CA, USA

    Eve J. Fine, Carl-Mikael Suomivuori & Ron O. Dror

  4. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA

    Eve J. Fine, Carl-Mikael Suomivuori & Ron O. Dror

  5. Institute for Computational and Mathematical Engineering, Stanford University, Stanford, CA, USA

    Eve J. Fine, Carl-Mikael Suomivuori & Ron O. Dror

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  1. Zheng He
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Contributions

Z.H., J.Z. and Y.X. carried out biochemical, structural and functional experiments. E.J.F., C.-M.S. and R.O.D. contributed to modelling. Z.H. and L.F. wrote the manuscript with input from all authors. L.F. directed the project.

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Correspondence to Liang Feng.

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Extended data figures and tables

Extended Data Fig. 1 Sequence alignment of MPC homologues.

a-b, Sequence alignment of MPC1 and MPC2 homologues. Secondary structural elements are indicated above the sequence alignment, while substrate and inhibitor binding residues are shown below the sequence alignment, represented by green and red circles, respectively.

Extended Data Fig. 2 Purification of MPC complex and structural comparison of MPC and SemiSWEET.

a, Size-exclusion chromatography profile of the heterodimer of MPC1 and MPC2 and SDS-PAGE analysis. The experiments were repeated independently six times with similar results. For gel source data, see Supplementary Fig. 1a. b, Size-exclusion chromatography profile of the complex of MPC-NbS1755-Legobody in detergent and SDS-PAGE analysis. The experiments were repeated independently six times with similar results. For gel source data, see Supplementary Fig. 1b. c, Cryo-EM density map of the complex of MPCUK5099-NbS1755-Legobody. MPC1, MPC2, NbS1755, Legobody, and nanodisc are colored in green, cyan, orang, gray, and purple, respectively. d, Structure comparison of hMPC and SemiSWEET.

Extended Data Fig. 3 Inhibitor and substrate binding and V74WMPC2 variant.

a, Chemical structures of select MPC inhibitors and their IC50 values. b-d, MPC’s binding pocket for UK5099, AKOS, and GW604714X. The slab view of MPC is shown (colored by electrostatic potential). e, Structural comparison of MPC in complex with pyruvate and UK5099. The pyruvate is colored in purple and UK5099 is colored in light pink. f-g, Docking of compound 2 and 12 (ref. 11) in MPC, compared to the experimental pose of AKOS. The AKOS is coloured in light orange, and the other inhibitors are coloured in gray. h, Effect of V74WMPC2 substitution on the yeast growth. The yeasts are grown on the synthetic defined medium that lacks leucine and valine (SD-L-V). WT, wild type; EV, empty vector. i-l, Known substrates (i, acetoacetate; j, beta-hydroxybutyrate; k, dichloroacetate; l, 2-chloroacetate) modelled in the substrate binding-pocket of MPC. Substrate molecules were placed in the pocket by aligning with the pyruvate. Pyruvate and other substrates are shown as sticks in purple and yellow, respectively. m, Structural comparison of MPC that was bound with UK5099 and GW604714X, respectively. The MPC in complex with UK5099 was shown in grey. For the MPC complexed with GW604714X, its two subunits, MPC1 and MPC2, are colored in green and cyan, respectively.

Extended Data Fig. 4 Cryo-EM data processing of MPCAKOS-NbS1755-Legobody.

a, A flowchart of MPCAKOS-NbS1755-Legobody data processing. b, A representative cryo-EM image (from 16,520 micrographs with similar results). c, Typical 2D class averages. d, A cut-open view of the local resolution map. e, Angular particle distribution. f, Gold-standard Fourier shell correlation curves of the local-refined map. g, Cryo-EM density and structural model.

Extended Data Fig. 5 Cryo-EM data processing of MPCUK5099-NbS1755-Legobody.

a, A flowchart of MPCUK5099-NbS1755-Legobody in nanodiscs data processing. This includes a cut-open view of the local resolution map, angular particle distribution and gold-standard Fourier shell correlation curves of the local-refined map. b, A representative cryo-EM image (from 19,801 micrographs with similar results). c, Typical 2D class averages. d, A flowchart of MPCUK5099-NbS1755-Legobody in LMNG data processing. e, Cryo-EM density and structural model.

Extended Data Fig. 6 Cryo-EM data processing of MPCpyruvate-NbS1755-Legobody.

a, A flowchart of MPCpyruvate-NbS1755-Legobody data processing. b, A representative cryo-EM image (from 4,775 micrographs with similar results). c, Typical 2D class averages. d, A cut-open view of the local resolution map. e, Angular particle distribution. f, Gold-standard Fourier shell correlation curves of the local-refined map. g, Cryo-EM density and structural model.

Extended Data Fig. 7 Cryo-EM data processing of MPCGW604714X-NbS1755-Legobody.

a, A flowchart of MPCGW604714X-NbS1755-Legobody data processing. b, A representative cryo-EM image (from 7,457 micrographs with similar results). c, Typical 2D class averages. d, A cut-open view of the local resolution map. e, Angular particle distribution. f, Gold-standard Fourier shell correlation curves of the local-refined map. g, Map vs. model FSC. h, Cryo-EM density and structural model.

Extended Data Fig. 8 Sensitivity of hMPC C54AMPC2 to inhibitors and comparison of inhibitor binding pockets between human MPC1/2 and yeast MPC1/3.

a, The sensitivity of the yeast growth to inhibitor UK5099 for yeasts expressing human MPC WT and C54AMPC2 (mean ± SD; n = 3 independent experiments). b, The sensitivity of the yeast growth to inhibitor AKOS for yeasts expressing human WT and C54AMPC2 (mean ± SD; n = 3 independent experiments). c, The residues that differ in the UK5099 binding pocket of hMPC and yMPC. The predicted yMPC (MPC1/MPC3) structure, modeled based on hMPC structure, was superimposed onto the hMPC structure. The differing residues in the inhibitor-binding pocket are shown as sticks in green (hMPC1), cyan (hMPC2), and purple (yMPC). d, The sensitivity of the yeast growth to inhibitor UK5099 for yeasts expressing WT MPC and mutants MPC (mean ± SD; n = 3 independent experiments).

Extended Data Fig. 9 Sequence alignment of MPC and SemiSWEET homologues.

Sequence alignments of MPC from human, Bovine, Mouse, Chicken, African clawed frog (frog), Black rockcod (Rockcod), Caenorhabditis elegans (C. ele), Lacerta muralis (L. mur), Saccharomyces cerevisiae (Yeast) and SemiSWEET from Vibrio sp (V. sp.), Rickettsia bellii (R. bel.), Leptospira biflexa serovar Patoc (L. bsp.), Fusobacterium (Fus.), Flavobacterium johnsoniae (F. joh.), Escherichia coli (E. coli), Chlorobium phaeobacteroides (C. pha.), Parvimonas micra (P. mic.), Limosilactobacillus reuteri (L. reu). The highly conserved residues between MPC2 and SemiSWEET are indicated by blue circles. Each group of MPC1, MPC2, and SemiSWEET is separated by lines.

Extended Data Table 1 Cryo-EM data collection, refinement, and validation statistics
Full size table

Supplementary information

Supplementary Fig. 1

Source images for SDS–PAGE gels in Extended Data Fig. 2a,b.

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He, Z., Zhang, J., Xu, Y. et al. Structure of mitochondrial pyruvate carrier and its inhibition mechanism. Nature 641, 250–257 (2025). https://doi.org/10.1038/s41586-025-08667-y

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