Structure of intact human MCU supercomplex with the auxiliary MICU subunits
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Protein & Cell
LETTER
Dear Editor, 2+
Mitochondrial Ca homeostasis regulates energy production, cell division, and cell death. The basic properties of mitochondrial Ca2+ uptake have been firmly established. The Ca2+ influx is mediated by MCU, driven by membrane potential and using a uniporter mechanism (Vasington and Murphy, 1962). Patch-clamp analysis of MCU currents demonstrated that MCU is a channel with exceptionally high Ca2+ selectivity (Kirichok et al., 2004). Even in the absence of structural data on the MCU complex, mitochondrial Ca2+ uptake and its regulation in mammals has been assumed to rely on a complex comprising MCU, EMRE, MICU1, and MICU2 (De Stefani et al., 2016). Previous models generally believe that MICU1 and MICU2 form a cap to occlude the MCU channel in low [Ca2+] condition, and when [Ca2+] is elevated, through conformational changes of the EF hands in these two regulators, they will depart from the MCU/EMRE pore to allow Ca2+ permeation (Phillips et al., 2019). In the present study, we tried to express the MCU complex in HEK 293F cells which were transfected by BacMam viruses for the genes mcu, mcub, micu1, micu2, and emre. After extensive optimization, we obtained an abundant amount of high quality human MCU-EMRE-MICU1-MICU2 supercomplex (MEMMS) protein samples, pulled-down by the C-terminally Strep-tagged EMRE. These samples were used to prepare grids for cryo-EM analyses (Fig. S1A, S1B and Methods). Images were recorded with a combination of a Titan Krios Cryo-EM and a K2 direct electron detector in super-resolution mode (Fig. S1C and S1D). After routine classification and refinement, further focused refinements for three regions were performed, and the three focused density maps were combined to generate an overall map of MEMMS at an improved resolution of 3.3–3.7 Å (gold-standard FSC 0.143 criterion) (Fig. S2). Compared to a most recent study (Fan et al., 2020), our MEMMS structure showed more accurate information about interactions between MICU1 and EMRE. Based on our structural and functional analysis, we conclude that MEMMS is an integral unit in mammals, EMRE may act as a lever to regulate the matrix gate of the MCU channel, and MICU1/2 enhance the Ca2+ uptake by interactions with the C- termini of EMRE in high [Ca2+] condition.
© The Author(s) 2020
MEMMS has a molecular weight of about 480 kDa and an overall dimension of 210 Å × 190 Å. The overall structure forms an O-shaped ring and adopts the shape like that of two “goldfish”, as if glued together at both their heads (MICU1/ MICU2 dimer) and tails (NTD of MCU). A pair of MICU1MICU2 heterodimer appears like a bridge across the gap between the two halves of MCU-EMRE complex (Fig. 1A). The well-resolved density map allowed us to build a structural model for almost all residues with their side chains (Figs. 1A, S3, and S4). However, three sets of densities were not optimal for model building. The first is the density for the highly conserved C-terminal poly-D tail (EMRE101−107: EDDDDDD) of EMRE, the second is the conserved N-terminal p
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