The mitochondrial calcium uniporter (MCU) is a highly selective calcium channel localized to the inner mitochondrial membrane. Here, we describe the structure of an MCU orthologue from the fungus Neosartorya fischeri (NfMCU) determined to 3.8 Å resolution by phase-plate cryo-electron microscopy. The channel is a homotetramer with two-fold symmetry in its amino-terminal domain (NTD) that adopts a similar structure to that of human MCU. The NTD assembles as a dimer of dimers to form a tetrameric ring that connects to the transmembrane domain through an elongated coiled-coil domain. The ion-conducting pore domain maintains four-fold symmetry, with the selectivity filter positioned at the start of the pore-forming TM2 helix. The aspartate and glutamate sidechains of the conserved DIME motif are oriented towards the central axis and separated by one helical turn. The structure of NfMCU offers insights into channel assembly, selective calcium permeation, and inhibitor binding.
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We thank J. Guo and Q. Chen for technical assistance and manuscript preparation, and J. Frauenfeld (Salipro Biotech) for the recombinant saposin construct. Negative-stained EM data acquisition and sample optimization for cryo-EM were performed at the University of California San Francisco; we thank M. Braunfeld for supervision of the microscopes. Phase plate cryo-EM data were collected at the University of Texas Southwestern Medical Center Cryo-EM Facility, which is funded by the CPRIT Core Facility Support Award RP170644; we thank D. Nicastro and Z. Chen for technical support and facility access. This work was supported in part by the Howard Hughes Medical Institute (to Y.J., V.K.M. and Y.C.), the National Institutes of Health (grant GM079179 to Y.J.; grants R01GM098672, S10OD020054, and S10OD021741 to Y.C.), the Welch Foundation (Grant I-1578 to Y.J.), the Cancer Prevention and Research Initiative of Texas (to X.B.) and the Virginia Murchison Linthicum Scholar in Medical Research fund (to X.B.).
Nature thanks M. Prakriya, D. Stokes and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, Representative gel filtration profile of full-length (black trace) versus truncated NfMCU used for cryo-EM studies (blue trace). b, Functional activity of full-length (FL) and truncated NfMCU (EM); data shown represent mean ± s.e.m. (n = 3 independent experiments). Cell lysates were analysed by immunoblotting using anti-His antibody to detect expression of NfMCU; RpoA was used as loading control. c, Detergent-solubilized NfMCU (control) separates as a single band at ~46 kDa on Coomassie-stained SDS–PAGE and crosslinks as tetramers when treated with chemical crosslinkers of varying spacer arm length. DSG, disuccinimidyl glutarate; DSS, disuccinimidyl suberate; BS3, bis(sulfosuccinimidyl) suberate; BSPEG5, PEGylated bis(sulfosuccinimidyl)suberate. See Supplementary Fig. 1 for gel source data. The experiments shown in a and b were repeated at least three times independently with similar results. The crosslinking experiment shown in c was performed once. Source Data
a, Calcium uptake in E. coli expressing empty vector (black trace) or NfMCU (blue trace), demonstrating that heterologously expressed NfMCU can confer uniporter function to bacteria. Arrows indicate addition of 20 µM CaCl2 to the reaction solution. b, Concentration-dependent calcium uptake in E. coli expressing empty vector (black trace) or NfMCU (blue trace). Each point represents the rate of calcium uptake normalized to the uptake rate at 90 µM CaCl2. c, Concentration-dependent inhibition of NfMCU by GdCl3 in E. coli expressing NfMCU. Each point represents the rate of calcium uptake normalized to the uptake rate without GdCl3 in the reaction. d, Concentration-dependent inhibition of Ca2+ uptake in E. coli expressing NfMCU by the MCU-specific inhibitor Ru360. Each point represents the rate of calcium uptake normalized to the uptake rate without Ru360 in the reaction. The Ru360 concentration required to reduce the Ca2+ uptake rate by half (IC50) is calculated to be 0.5 μM. e, In light of the contradiction of the oligomeric state between NfMCU and cMCU-∆NTD, we also designed an in vitro functional assay by reconstituting our purified tetrameric channel into liposomes. Unable to obtain currents of NfMCU by electrophysiology—probably owing to the channel’s small conductance—we performed a radioactive 45Ca flux assay on proteoliposomes loaded with 150 mM KCl. Shown here is the time-dependent 45Ca2+ uptake by empty liposomes or NfMCU proteoliposomes incubated with 45Ca2+ in a reaction buffer containing 150 mM KCl (red points) or 150 mM NMDG (blue points). The result demonstrates that in a reaction solution containing 150 mM NMDG and the K+-selective ionophore valinomycin (to generate an electrical driving force), NfMCU proteoliposomes exhibited time-dependent 45Ca2+ uptake while the same proteoliposomes in a reaction solution containing valinomycin and 150 mM KCl (to eliminate electrical driving force) accumulated 45Ca2+ only slightly better than empty liposomes, recapitulating the voltage-dependent Ca2+ uptake property of the MCU. f, Concentration-dependent inhibition of NfMCU proteoliposomes by Ru360, demonstrating that Ru360 also blocks NfMCU 45Ca2+ uptake in a concentration-dependent manner up to 50%, which is expected given an equal distribution of two channel orientations in the liposomes. Each point represents radioactivity measured after 30 min reaction normalized to sample without Ru360. All data in a–f are shown as mean ± s.e.m. (n = 3 independent experiments). Source Data
a, Representative phase-plate electron micrograph of the NfMCU saposin complex and 2,470 micrographs were used for structure determination. b, Representative 2D class averages of the NfMCU saposin complex. c, Flowchart of data processing to obtain the 3.8 Å cryo-EM map of the NfMCU saposin complex. Details can be found in the Image processing section of the Methods. d, Local resolution of NfMCU estimated with RELION2.0. e, FSC curves for the refined model versus the summed 3.8 Å map (black curve), the refined model versus half map 1 (red curve), and the refined model versus half map 2 that was not used for refinement (green curve). f, The gold-standard FSC curve for the cryo-EM map.
a, Representative regions of the EM map of NfMCU highlighting key structural features at the NTD (α1, α2, β2– β3), the coiled-coil domain, and transmembrane domain (TM1 and TM2). Protomers 1 and 2 are shown in yellow and orange, respectively. b, Stereo view of the EM map carved around the TM2 filter region of NfMCU.
a, Cryo-EM map of NfMCU in complex with saposin (shown alternating between purple and pink). The map is low-pass filtered to 5 Å to allow better visualization of the density from the saposin molecules. Six saposin molecules, each oriented at a 45° angle to the vertical axis, wrap around NfMCU stabilized in lipid. b, Ribbon diagram of NfMCU in complex with saposin. Approximately 79–81 residues of the 81-residue saposin molecule were modelled as a poly-alanine chain into the helix–turn–helix EM map density for saposin.
The sequences were aligned using PROMALS3D50 and numbered according to NfMCU. Secondary structures shown above the sequences are based on the cryo-EM structure of NfMCU. Loops are denoted by solid black lines; disordered regions not observed in the cryo-EM structure and truncations made to NfMCU are indicated by dashed lines. The first 75 residues in NfMCU (grey arrow) are predicted to be the mitochondrial targeting sequence (MTS). NCBI accession number for the sequences are: HsMCU (human MCU): NP_612366.1, MmMCU (mouse MCU): XP_006513531.1, DdMCU (D. discoideum MCU): XP_637750.1, CeMCU (C. elegans MCU): NP_500892.1, NcMCU (Neurospora crassa MCU): XP_959658.1, NfMCU (Neosartorya fischeri MCU): XP_001266985.1.
a, Overall structure comparison between tetrameric NfMCU and pentameric cMCU-∆NTD (PDB: 5ID3). Each subunit is individually coloured. b, Structure comparison between a single subunit of the pore domain of NfMCU and cMCU-∆NTD. Dashed boxes indicate major differences between the two structures. Structural features in cMCU-∆NTD are labelled as in the original structure16. IJMH, inner juxtamembrane helix; OJMH, outer juxtamembrane helix; L1, loop 1; L2, loop 2; JML, juxtamembrane loop; CC1, coiled-coil 1; CC2, coiled-coil 2. c, Structural comparison between the NTDs of NfMCU and HsMCU (PDB: 4XTB). Dashed box indicates the major difference between the two structures. d, Atomic interactions between two NTD subunits in the crystal structure of HsMCU NTD, equivalent to interface I identified in the cryo-EM structure of NfMCU.
NfMCU, with its mitochondrial targeting sequence removed and replaced with a 6×His tag, was expressed in E. coli for functional analysis. Mouse anti-His antibodies (Qiagen) were used to detect NfMCU expression and mouse anti-RpoA targeting E. coli RNA polymerase alpha (Santa Cruz Biotechnology) was used for loading control. See Supplementary Fig. 1 for gel source data. The experiment was repeated three times independently with similar results.
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Journal of The American Society for Mass Spectrometry (2019)
Nature Reviews Molecular Cell Biology (2018)