Structural and mechanistic insights into MICU1 regulation of mitochondrial calcium uptake

Abstract

Mitochondrial calcium uptake is a critical event in various cellular activities. Two recently identified proteins, the mitochondrial Ca(2+) uniporter (MCU), which is the pore-forming subunit of a Ca(2+) channel, and mitochondrial calcium uptake 1 (MICU1), which is the regulator of MCU, are essential in this event. However, the molecular mechanism by which MICU1 regulates MCU remains elusive. In this study, we report the crystal structures of Ca(2+)-free and Ca(2+)-bound human MICU1. Our studies reveal that Ca(2+)-free MICU1 forms a hexamer that binds and inhibits MCU. Upon Ca(2+) binding, MICU1 undergoes large conformational changes, resulting in the formation of multiple oligomers to activate MCU. Furthermore, we demonstrate that the affinity of MICU1 for Ca(2+) is approximately 15-20 μM. Collectively, our results provide valuable details to decipher the molecular mechanism of MICU1 regulation of mitochondrial calcium uptake.

Figure 1

Crystal structures of Ca²⁺-free and Ca²⁺-bound human MICU1. A A schematic drawing of human MICU1. B, C Cartoon representation of the overall structure of MICU1 in the Ca²⁺-free and the Ca²⁺-bound state. The N-domain, N-lobe, C-lobe, C-helix, and Ca²⁺ are colored cyan, orange, green, purple, and red, respectively. D Structural comparisons of four helix-loop-helix structural units before and after Ca²⁺ binding. The Ca²⁺-free form is colored yellow, and the Ca²⁺-bound form is colored blue. The calcium ions are shown as red spheres.

Figure 2

The C-helix is critical for mitochondrial calcium uptake. A, B Multi-angle laser scattering analysis of MICU1-xtal in the absence or presence of Ca²⁺. MALS-determined molecular weights were calculated over prominent peaks (red line). C The hexameric packing of Ca²⁺-free MICU1. The surface map of six molecules in the asymmetric unit is shown. A cartoon model of the C-helix is presented in the center of the hexamer. D The anomalous scattering map of the selenium-substituted Ca²⁺-free MICU1-xtal structure. The map colored in magenta was contoured at 3.0σ. Cartoon representations of the five C-helices from different Ca²⁺-free MICU1 molecules are shown. One C-helix is not included in the final model due to poor electron density. The methionines are depicted as sticks, and Se atoms are depicted as spheres. E, F Multi-angle laser scattering analysis of MICU1-xtal-deltaC in the absence or presence of Ca²⁺. G Sequence alignment of the MICU1 protein from human (Q9BPX6), bovine (Q0IIL1), mouse (Q8VCX5), chick (E1BWC6), fish (A4IG32), and frog (B1H2N3). The residues that are conserved among the species are highlighted in red. The residue numbers of human MICU1 are shown in black. H Western blot analysis of co-immunoprecipitated wild-type FL-MICU1 and mutant DeltaC-MICU1 with the MCU channel in the absence of Ca²⁺. I cDNA rescue of the RNAi mitochondrial calcium uptake phenotype. Source data are available online for this figure.

Figure 3

The MICU1 dimer. A, B A cartoon representation of the MICU1 dimer in the absence or presence of Ca²⁺. Each molecule in one dimer is colored differently. Calcium is shown as a red sphere. C Superposition of Ca²⁺-free and Ca²⁺-bound MICU1. The Ca²⁺-bound MICU1 dimer is colored green. One molecule of Ca²⁺-free MICU1 (orange) superposed well with Ca²⁺-bound MICU1. However, the second molecule of Ca²⁺-free MICU1 (red) required a 74° rotation for superposition onto Ca²⁺-bound MICU1. D, E Detailed interactions within interfaces of Ca²⁺-free and Ca²⁺-bound MICU1 dimers. Each molecule in the dimer is colored green and orange, respectively. The oxygen and nitrogen atoms are colored red and blue, respectively. Hydrogen bonds are depicted as red dotted lines. F, G Sedimentation velocity analysis of the MICU1-xtal-deltaC wild-type and mutants in the absence or presence of Ca²⁺. H cDNA rescue of the RNAi mitochondrial calcium uptake phenotype.

Figure 4

MICU1 calcium-binding sites. A, B Detailed interactions of two calcium-binding sites of Ca²⁺-bound MICU1. Calcium ions are colored yellow. The oxygen and nitrogen atoms are colored red and blue, respectively. A water molecule is denoted as “wat.” The side chains of several residues are not shown for clarity. C, D The dissociation constants of MICU1-xtal-deltaC mutants (D421A-E432K and D231A-E242K) in the presence of calcium ions were determined using ITC.