Abstract
The mitochondrial pyruvate carrier (MPC) is a mitochondrial inner membrane protein complex that is essential for the uptake of pyruvate into the mitochondrial matrix as the primary carbon source for the tricarboxylic acid cycle1,2. Here we present six cryo-electron microscopy structures of human MPC in three states: three structures in the intermembrane space (IMS)-open state, obtained in different conditions; a structure of pyruvate-treated MPC in the occluded state; and two structures in the matrix-facing state, bound with the inhibitor UK5099 or with an inhibitory nanobody on the matrix side. MPC is a heterodimer consisting of MPC1 and MPC2, with the transmembrane domain adopting pseudo-C2 symmetry. Approximate rigid-body movements occur between the IMS-open state and the occluded state, whereas structural changes, mainly on the matrix side, facilitate the transition between the occluded state and the matrix-facing state, revealing an alternating access mechanism during pyruvate transport. In the UK5099-bound structure, the inhibitor fits well and interacts extensively with a pocket that opens to the matrix side. Our findings provide key insights into the mechanisms that underlie MPC-mediated substrate transport, and shed light on the recognition and inhibition of MPC by UK5099, which will facilitate the future development of drugs that target MPC.
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Data availability
Atomic coordinates and corresponding electron microscopy maps of the structures of apo-MPC in complex with Nb1 at pH 8.0 (Protein Data Bank (PDB) ID: 8YW6 and Electron Microscopy Data Bank (EMDB) ID: EMD-39624) and pH 6.8 (PDB ID: 9KNW and EMDB ID: EMD-62464) in the IMS-open state; the structure of pyruvate-treated MPC in complex with Nb1 at pH 8.0 in the IMS-open state (PDB ID: 9KNY and EMDB ID: EMD-62466); the structure of pyruvate-treated MPC in complex with Nb1 at pH 6.8 in the occluded state (PDB ID: 9KNX and EMDB ID: EMD-62465); the structure of pyruvate-treated MPC in complex with Nb1 and Nb2 at pH 6.8 in the matrix-facing and inhibitory state (PDB ID: 8YW9 and EMDB ID: EMD-39626); and the UK5099-bound structure (PDB ID: 8YW8 and EMDB ID: EMD-39625) have been deposited in the PDB (http://www.rcsb.org) and the EMDB (https://www.ebi.ac.uk/pdbe/emdb/), respectively. The SemiSWEET structures used for structural comparisons with MPC in Extended Data Fig. 9 can be accessed through the PDB with IDs 4X5M, 4RNG, 4QNC and 4QND. Source data are provided with this paper.
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Acknowledgements
We thank the following facilities at Westlake University: the cryo-EM facility for providing cryo-EM support; the High-Performance Computing Center for computational resources and related assistance; and the radioisotope laboratory for providing related facilities and assistance. We thank Q. Hu for discussions about the project and help revising the manuscript draft. This work was supported by the Ministry of Science and Technology of China, National Key R&D Program (project 2022YFA1303700); the Key R&D Program of Zhejiang (2024SSYS0029); and the Westlake Education Foundation.
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Contributions
D.M. conceived the project and wrote the manuscript. D.M. supervised and provided methodology for the structural and biochemical studies of MPC. X.W. supervised and provided methodology for the nanobody screening, validation and application. J.L. contributed to protein expression and purification, cryo-EM sample preparation, data collection, assay system set-up and biochemical assays. J.S. contributed to cryo-EM data processing, model building, purification of mutants and binding assays. A.S. contributed to nanobody screening, validation and purification. M.L. and M.X. contributed to homologue screening and assay system set-up. K.Z. and P.L. performed ligand docking. G.H. contributed to cryo-EM data processing.
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Extended data figures and tables
Extended Data Fig. 1 Cryo-EM sample preparation and validation.
a, Time-course profile for pyruvate transport by purified WT MPC. Black dots represent readings for proteoliposomes incorporated with MPC and black squares represent readings for empty liposomes. Internal pH was 8.0 and external pH was 6.4. In the following transport assay experiments we chose to measure pyruvate uptake by MPC at the time point of 30 s, which is approximately within the linear range. b, Influence of different termination conditions on substrate transport. In the following transport assay experiments we added 1 mM of UK5099 for transport termination. c, Km value determination for WT MPC. The Km value was obtained from automatic curve fitting within the pyruvate concentration range of 100 to 600 μM. d, Transport activity of WT MPC with or without binding to nanobodies. MPC samples were pre-incubated with corresponding nanobodies before incorporated into liposomes. Liposomes were run into SDS–PAGE to monitor incorporation efficiency of different MPC samples. e, Protein complexes purified for cryo-EM analyses. Peak containing desired complex in each gel filtration profile is indicated by a red star, and peak fractions were analysed by SDS–PAGE. Each MPC sample was purified at least twice with similar behaviour. For all gels, the same molecular weight marker was used as that in d, and for uncropped gels, see Supplementary Fig. 1. In all transport assay figures, data are shown as mean ± s.d., n = 3 technical replicates. All transport assay experiments were independently repeated twice with similar results.
Extended Data Fig. 2 Cryo-EM analyses of MPC in the IMS-open state.
a, Cryo-EM analysis of apo-MPC in complex with Nb1 at pH 8.0. b, Cryo-EM analysis of apo-MPC in complex with Nb1 at pH 6.8. c, Cryo-EM analysis of pyruvate-treated MPC in complex with Nb1 at pH 8.0. For each sample, a representative raw micrograph, 2D-class averages of particle images, the flow chart of the EM analysis, the local-resolution map, the angular distribution of the particles, the Fourier shell correlation (FSC) curve and the FSC curve of the refined model versus the corresponding map in MPC region are shown.
Extended Data Fig. 3 Cryo-EM analyses of MPC in the occluded state and in the matrix-facing state.
a, Cryo-EM analysis of pyruvate-treated MPC in complex with Nb1 at pH 6.8. b, Cryo-EM analysis of UK5099-bound MPC in complex with Nb1. c, Cryo-EM analysis of pyruvate-treated MPC in complex with Nb1 and Nb2 at pH 6.8. For each sample, a representative raw micrograph, 2D-class averages of particle images, the flow chart of the EM analysis, the local-resolution map, the angular distribution of the particles, the Fourier shell correlation (FSC) curve and the FSC curve of the refined model versus the corresponding map in MPC region are shown.
Extended Data Fig. 4 Structural features of MPC structures in the IMS-open state.
a, Overall structures of MPC purified in different conditions that exhibit IMS-open conformation. b, Structural alignment of the three MPC structures in IMS-open state. c, Superimposition of TMDs of MPC1 and MPC2. d, Structural details of MPC2-APH. e, Density of the cardiolipin molecule found between MPC2-APH and MPC1-TMD. f, Density of the PC molecule found between MPC1-TM1 and MPC2-TM2. All figures of local densities were generated by isomesh function in PyMOL at sigma value of 4.5.
Extended Data Fig. 5 Importance of structural features in the occluded MPC structure.
a, Cryo-EM densities of residues forming the putative pyruvate-binding pocket in the occluded MPC structure. b, Gel filtration profiles of corresponding MPC variants. Peak positions of MPC are indicated by reverse triangles. The differences in elution volume of MPC variants are mainly due to different equipment and columns were used during purification. Each MPC mutant was purified at least twice with similar behaviour. c, Substrate transport by MPC carrying mutations on residues in the putative pyruvate-binding pocket. d, Time courses of pyruvate transport by putative pyruvate-binding-pocket mutants. e, Cryo-EM densities of the L1 loops in the occluded structure. f, Substrate transport by MPC carrying mutations on residues in the L1 loops. g, Cryo-EM densities of residues mediating interaction between MPC1 and MPC2 below the L1 loops on the IMS side of the occluded structure. h, Substrate transport by MPC carrying MPC2-Q71A, Q110A and Q110H mutations. For all gels, the same molecular weight marker was used as that in c, and for uncropped gels, see Supplementary Fig. 1. In all transport assay figures, data are shown as mean ± s.d., n = 3 technical replicates. All transport assay experiments were independently repeated twice with similar results.
Extended Data Fig. 6 Binding features of inhibitors towards MPC.
a, Chemical structure of UK5099. Key structural elements of UK5099 discussed in main text are indicated. b, Densities of UK5099 and binding-pocket-forming residues. All densities are isolated at sigma value of 4.5. c, Gel filtration profiles of WT MPC and the mutant carrying MPC2-W82D mutation. The mutant was purified at least twice with similar behaviour. d, Microscale thermophoresis (MST) binding measurement of MPC variants towards UK5099 at different maximum UK5099 concentrations. Binding curves of MPC variants are presented. Binding affinity between WT and UK5099 is 353.8 ± 97.03 nM, while binding between binding-pocket mutants and UK5099 was not detectable in the UK5099 concentration range of up to 2 μM. Binding affinities between the mutants and UK5099 could be detected at 159.2 ± 26.72 μM for K49A mutant, 122.8 ± 20.57 μM for W82A mutant, 269.1 ± 15.40 μM for W82D mutant and 122.4 ± 22.99 μM for N100A mutant, respectively, in the UK5099 concentration range of up to 300 μM. Error bars represent mean ± s.d. based on three independent measurements. e, Substrate transport of WT MPC and the mutant carrying MPC2-W82D mutation. Data are shown as mean ± s.d., n = 3 technical replicates. Experiments were independently repeated twice with similar results. For uncropped gel, see Supplementary Fig. 1. f, Ligand docking results of known inhibitors towards MPC. General fitting of inhibitors in the UK5099-binding pocket in the overall structure context of MPC. g, In silico docking results of known inhibitors targeting the UK5099-binding pocket. Ligands are shown in the UK5099-binding pocket (grey, 60% transparency).
Extended Data Fig. 7 Cryo-EM densities of the putative gating residues on the matrix side.
Densities of corresponding residues in the occluded structure and the Nb2-bound matrix-facing structure are shown.
Extended Data Fig. 8 Sequence alignments of MPC subunits among different species.
a, Sequence alignment of MPC1. Sequences were obtained from UniProt with the following accession codes of MPC1 from corresponding species: Homo sapiens (Q9Y5U8), Mus musculus (P63030), Saccharomyces cerevisiae (P53157), Caenorhabditis elegans (Q21828), Drosophila melanogaster (Q7KSC4) and Danio rerio (F1Q6Z3). b, Sequence alignment of MPC2 from different species and MPC3 from S. cerevisiae. Sequences were obtained from UniProt with the following accession codes of MPC2 from corresponding species: H. sapiens (O95563), M. musculus (Q9D023), S. cerevisiae (P38857), C. elegans (O01578), D. melanogaster (Q9VHB2), D. rerio (Q7ZUJ3) and MPC3 from S. cerevisiae (P53311). In the sequence alignment results in a and b, residues forming the putative pyruvate-binding pocket in the occluded structure are indicated by reversed blue triangles, zipper-forming residues on L1 loops are indicated by reversed red triangles, residues mediating interactions between MPC1 and MPC2 below L1 loops are indicated by green squares, and UK5099-binding-pocket-forming residues are indicated by red dots. c, Difference in UK5099-binding pocket between human MPC and yeast MPC. Pockets were generated using PyMOL. The pocket of yeast MPC was mimicked by mutating corresponding residues in our UK5099-bound cryo-EM structure model. The opening of each binding pocket is highlighted by a red dashed line.
Extended Data Fig. 9 Structural comparison between structures of MPC and SemiSWEETs.
a, Structural comparison between the IMS-open MPC and an inward-open (cytoplasm-facing) SemiSWEET (PDB ID: 4X5M). b, Structural comparison between occluded structure of MPC and structures of SemiSWEETs (PDB IDs: 4RNG and 4QNC) in occluded conformation. c, Structural comparison between the matrix-facing MPC and an outward-open SemiSWEET (PDB ID: 4QND). d, The putative substrate-binding pocket in SemiSWEET. An occluded structure of SemiSWEET (PDB ID: 4RNG) was used for the cut-away surface generation. e, Comparison between the putative substrate-binding-pocket-forming residues in occluded structures of MPC and SemiSWEET.
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Liang, J., Shi, J., Song, A. et al. Structures and mechanism of the human mitochondrial pyruvate carrier. Nature 641, 258–265 (2025). https://doi.org/10.1038/s41586-025-08873-8
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DOI: https://doi.org/10.1038/s41586-025-08873-8
