Abstract

Mitochondrial calcium uptake is critical for regulating ATP production, intracellular calcium signalling, and cell death. This uptake is mediated by a highly selective calcium channel called the mitochondrial calcium uniporter (MCU). Here, we determined the structures of the pore-forming MCU proteins from two fungi by X-ray crystallography and single-particle cryo-electron microscopy. The stoichiometry, overall architecture, and individual subunit structure differed markedly from those described in the recent nuclear magnetic resonance structure of Caenorhabditis elegans MCU. We observed a dimer-of-dimer architecture across species and chemical environments, which was corroborated by biochemical experiments. Structural analyses and functional characterization uncovered the roles of key residues in the pore. These results reveal a new ion channel architecture, provide insights into calcium coordination, selectivity and conduction, and establish a structural framework for understanding the mechanism of mitochondrial calcium uniporter function.

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Acknowledgements

We thank the staff at Beamline 24ID-C/E, 23ID-B/D, and 17ID (APS, Argonne National Laboratory), 12-2 (SSRL, SLAC National Laboratory), and 5.0.2 (Advanced Light Source). We thank Z. Li at HMS and C. Xu and K. Song at UMass cryo-EM facility for help with EM data collection. This work was made possible by support from Stanford University, the Klingenstein-Simons Fellowship, and the Harold and Leila Y. Mathers Charitable Foundation to L.F., and from a National Science Foundation Graduate Research Fellowship to N.M.F. NE-CAT is supported by NIH P41 GM103403 and S10 RR029205. We thank A. Manglik for discussions and R. Lewis for suggestions and comments.

Reviewer information

Nature thanks M. Prakriya, D. Stokes and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

    • Xiaofang Xu

    Present address: Department of Ophthalmology, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai, China

  1. These authors contributed equally: Chao Fan, Minrui Fan, Benjamin J. Orlando, Nathan M. Fastman

Affiliations

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

    • Chao Fan
    • , Minrui Fan
    • , Nathan M. Fastman
    • , Jinru Zhang
    • , Yan Xu
    • , Xiaofang Xu
    •  & Liang Feng
  2. Department of Cell Biology, Harvard Medical School, Boston, MA, USA

    • Benjamin J. Orlando
    • , Melissa G. Chambers
    •  & Maofu Liao
  3. Biophysics Program, Stanford University, Stanford, CA, USA

    • Nathan M. Fastman
    •  & Liang Feng
  4. NE-CAT and Dept. of Chemistry and Chemical Biology, Cornell University, Argonne National Laboratory, Argonne, IL, USA

    • Kay Perry

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Contributions

C.F. and M.F. performed protein purification, biochemistry, crystallization, data collection, crystallography and functional assays. B.J.O. performed negative-stain EM, cryo-EM, and data processing. N.M.F. performed protein engineering, crystallization, and data collection. Y.X. performed protein purification and biochemistry. J.Z. performed crystallography. M.G.C. helped with initial EM screening. X.X. helped with initial characterization. K.P. assisted with crystallography. M.L. oversaw the EM studies and contributed to EM data collection and processing. L.F. conceived the project, oversaw the X-ray structural, biochemical, and functional studies and contributed to experimental work and crystallography. L.F. and M.L. wrote the manuscript with input from all co-authors.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Maofu Liao or Liang Feng.

Extended data figures and tables

  1. Extended Data Fig. 1 Sequence alignment, sample preparations and functional characterizations.

    a, Sequence alignment of MCU. The protein sequences of MCUs from Fusarium graminearum (FgMCU), Metarhizium acridum (MaMCU), Homo sapiens (HsMCU) and Caenorhabditis elegans (CeMCU) were aligned using Clustal Omega with manual adjustment. The domain organization is shown above the sequences. b, FgMCU in DDM on size-exclusion chromatography. c, MaMCU in DDM on size-exclusion chromatography. Data in b, c are representative of five independent experiments with similar results. d, Ca2+ uptake activity of FgMCU in E. coli. Fluorescence changes upon addition of 0.5 mM Ca2+ are shown for wild-type (red), D351A/E354A (green), control (black), and with ruthenium red (blue). e, Cation flux activity of FgMCU in reconstituted liposomes. Fluorescence changes are shown for FgMCU (red) and with Gd3+ (blue). Data in d, e are representative of three independent experiments with similar results.

  2. Extended Data Fig. 2 Crystal packing and molecule interactions in the crystal.

    a, The crystal lattice structure of MaMCU-Rub. One MaMCU channel is coloured blue and its associated rubredoxin is coloured cyan. In the crystal, the transmembrane domain lacks clear protein-mediated contacts. Presumably, relatively disordered protein regions or detergent micelles mediate the contact. b, The crystal lattice structure of MaMCU in complex with nanobody. One MCU is shown in blue and the bound nanobodies are shown in cyan. c, The crystal lattice structure of the MaMCU soluble domain, shown in ribbons. Each chain is coloured differently. Two helices that formed a coiled-coil were derived from two different chains, in a domain-swapped manner. d, Close-up view of the interaction between nanobody and McMCU in the crystal. Nanobody binds near the end of TM1 and is distant from the selectivity filter (shown as coloured sticks). Nanobody is coloured grey and MaMCU is colored cyan. e, Mercury and selenium sites identified by MR-SAD. Two protomers of MaMCU in one asymmetric unit are shown as ribbons in green and blue, respectively. The mercury sites identified from ‘marker mutants’ are shown as red spheres; one selenium site from the labelled wild-type protein is shown in the same manner. f, 2FoFc electron density map of MaMCU (cyan mesh, 1σ) with the structure model shown in ribbon. g, Close-up view of 2FoFc electron density map of individual transmembrane helices (cyan mesh, 1.5σ) with side chains shown in sticks.

  3. Extended Data Fig. 3 Comparison of the NMR structure of cMCU with the crystal structure of MaMCU, and the structure of the soluble domain of MaMCU.

    a, Left, pentameric NMR structure of cMCU. Right, dimer-of-dimers structure of MaMCU. Box outlines the equivalent region including the transmembrane domain and coiled-coil. b, Comparison of a subunit of cMCU by NMR with that of MaMCU by crystallography. The helical part of the pore-lining TM2 is superimposed. The rest of the protein shows markedly different structure. Right, expanded view of the selectivity filter region. The most conserved WDxxEP residues (shown as sticks) are organized differently in these two structures. c, Ribbon representation of the soluble domain of MaMCU. Each subunit is coloured in blue, green, gold, or cyan, with the portions before and after the transmembrane domain of each subunit in slightly different colours. d, Structural comparison of the NTDs of MaMCU (purple) and human MCU (cyan). The two structures have the same fold and superimpose well onto each other. e, A pair of human MCU NTDs (cyan and blue) superimposes well onto one of dimers formed by MaMCU NTDs (purple and pink).

  4. Extended Data Fig. 4 Negative-stain EM and cryo-EM of MaMCU and FgMCU.

    a, Representative negative-stain EM image and 2D averages of MaMCU in A8–35 (from 28 micrographs with similar results), MaMCU in nanodiscs (from 28 micrographs with similar results), FgMCU in PMAL-C8 (from 20 micrographs with similar results), and FgMCU in nanodiscs (from 40 micrographs with similar results). The box dimension of the 2D averages is 215 Å. b, Representative cryo-EM image (from 1,862 micrographs with similar results), 2D averages, and image processing workflow of MaMCU in A8–35. The box dimension of the 2D averages is 172 Å. c, Representative cryo-EM image (from 4,055 micrographs with similar results), 2D averages, and image processing workflow of FgMCU in PMAL-C8. The box dimension of the 2D averages is 176 Å. d, Different views of the cryo-EM maps of MaMCU in A8–35 and FgMCU in PMAL-C8, both low-pass filtered to 7 Å. 2D averages and map calculation were not repeated.

  5. Extended Data Fig. 5 Single-particle cryo-EM analysis of FgMCU in nanodiscs.

    a, Representative cryo-EM image (from 5,332 micrographs with similar results). b, 2D averages of cryo-EM particle images. The box dimension is 187 Å. c, Image processing workflow. Two cryo-EM data sets were first independently subjected to 3D classification without symmetry, and the particle images in good classes were combined and further processed with two-fold symmetry, generating a 4.9 Å-resolution cryo-EM map. Subsequently, a final round of 3D classification focused on the transmembrane domain sorted out two major classes, which generated two final maps at 5.0 and 4.8 Å resolution for the transmembrane domain. The conformations in these two maps are designated as type 1 and type 2. 2D averages and map calculations were not repeated.

  6. Extended Data Fig. 6 Cryo-EM maps of two conformations of FgMCU in nanodiscs.

    a, Gold-standard FSC curves of the type 1 final cryo-EM map, calculated with a soft mask to include only the transmembrane domain (TMD, solid line) or the entire MCU molecule without nanodisc (dashed line). The estimated resolutions based on an FSC = 0.143 criterion are indicated. b, Cross-sectional view of the type 1 final cryo-EM map filtered to 5.0 Å resolution and coloured according to local resolution. c, Angular distribution of the particle images used for calculating the type 1 final cryo-EM map. df, Same as ac, but for the type 2 conformation. g, Two cross-sectional views of the transmembrane domains of the type 1 and type 2 cryo-EM maps, superimposed onto an MaMCU-based homology model of FgMCU. The missing density and extra densities in the type 2 map are indicated. Map calculations in a and d were not repeated.

  7. Extended Data Fig. 7 Tetrameric assembly of MaMCU, overall architecture of a metazoan MCU and symmetry mismatch of MaMCU.

    a, The design of cysteine pair mutations for crosslinking transmembrane subunits. L314 on TM1 and M348 on TM2 were mutated to cysteines (side chains shown as sticks). b, Site-directed disulfide cross-linking of MaMCU in detergent solution. L314 on TM1 and M348 on TM2 were mutated to create a cysteine pair at the protomer interface on an otherwise cysteine-free background. The double cysteine mutant was purified and oxidized by copper phenanthroline (CuP) for various time durations. The cross-link product was analysed by SDS–PAGE. c, Site-directed disulfide cross-linking of MaMCU in the membrane. Isolated membrane from the MaMCU L314C/M348C mutant was treated with CuP for an increasing amount of time. The cross-linking product was analysed by western blot using an antibody against the His tag on the protein. d, The size-exclusion profile of MaMCU with or without crosslinking. MaMCU L314C/M348C mutant treated or untreated with CuP in the membrane was purified and analysed by size-exclusion chromatography. The peak fractions were subjected to SDS–PAGE and visualized by Coomassie blue staining. e, Site-directed disulfide cross-linking of mosquito MCU in detergent solution. A pair of residues (T294 on TM1 and V341 on TM2) that were equivalent to those used in MaMCU crosslinking at the protomer interface were mutated to cysteine. The purified double cysteine mutant was oxidized by CuP and analysed by SDS–PAGE. The tetramer position on the SDS–PAGE gel is the same as that of MaMCU in b. f, 2D averages of negatively stained FgMCU in nanodiscs, mosquito MCU (T294C/V341C) in nanodiscs, and mosquito MCU (T294C/V341C) in amphipol A8–35. 2D averages were not repeated. g, Geometry of markers in transmembrane domain and NTD of MaMCU. Site-specific mercury marker on transmembrane domain (red spheres) and Cα position of Ile115 in NTD (blue spheres) are indicated. Data in be are representative of three independent experiments with similar results. For gel source data, see Supplementary Fig. 1.

  8. Extended Data Fig. 8 Expression levels of MaMCU mutants and ruthenium red sensitivity of selected mutants.

    a, The expression level of wild-type MaMCU and those with a point mutation in the transmembrane domain. Green fluorescent protein-tagged protein was analysed on SDS–PAGE and the protein was visualized by in-gel fluorescence. The experiments were repeated twice independently with similar results. For gel source data, see Supplementary Fig. 1. bm, The effect of substituting residues near the intermembrane surface of MaMCU on its ruthenium red sensitivity. Ca2+ uptake activity of the wild-type or mutant MaMCU was assayed in an E. coli-based assay (0.5 mM Ca2+, 1 μM RuR). The Ca2+ uptake in the absence (blue) or in the presence (black) of ruthenium red is reflected by fluorescence changes. The experiments were repeated three times independently with similar results.

  9. Extended Data Table 1 Statistics of X-ray data collection and refinement
  10. Extended Data Table 2 Cryo-EM data collection statistics

Supplementary information

  1. Supplementary Figure 1

    Source images for gel electrophoresis in Extended Data Figures 7 and 8.

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https://doi.org/10.1038/s41586-018-0330-9

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