Abstract
The mitochondrial calcium uniporter (MCU) plays a critical role in the mitochondrial calcium uptake into the matrix. In metazoans, the uniporter is a tightly regulated multi-component system including the pore-forming subunit MCU and several regulators (MICU1, MICU2, EMRE). The calcium-conducting activity of metazoan MCU requires the single-transmembrane protein EMRE. Dictyostelium discoideum (Dd), however, developed a simplified uniporter for which the pore-forming MCU (DdMCU) alone is necessary and sufficient for calcium influx. Here, we report a crystal structure of the N-terminal domain (NTD) of DdMCU at 1.7 Å resolution. The DdMCU-NTD contains four helices and two strands arranged in a fold that is completely different from the known structures of other MCU-NTD homologs. Biochemical and biophysical analyses of DdMCU-NTD in solution indicated that the domain exists as oligomers, most probably as a pentamer or hexamer. Mutagenesis showed that the acidic residues Asp60, Glu72 and Glu74, which appeared to mediate the parallel interface as observed in the crystal structure, participated in the self-assembly of DdMCU-NTD. Intriguingly, the oligomeric complex readily dissociated to lower-order oligomers in the presence of calcium. We propose that the calcium-triggered dissociation of NTD regulates the channel activity of DdMCU by a yet unknown mechanism.
Abbreviations
- BN-PAGE
- blue native polyacrylamide gel electrophoresis
- Ce
- Caenorhabditis elegans
- CHES
- 2-(cyclohexylamino)ethanesulfonic acid
- CMC
- critical micelle concentration
- Dd
- Dictyostelium discoideum
- Fg
- Fusarium graminearum
- Ma
- Metarhizium acridum
- Nc
- Neurospora crassa
- Cy
- Cyphellophora europaea
- Nf
- Neosartorya fischeri
- DLS
- dynamic light scattering
- DTT
- dithiothreitol
- EDTA
- ethylenediaminetetraacetic acid
- EMRE
- essential MCU regulator
- HEPES
- (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- Hs
- Homo sapiens
- IPTG
- isopropyl-1-thiogalactopyranoside
- LB
- Luria-Bertani
- MALS
- multi-angle light scattering
- MCU
- mitochondrial calcium uniporter
- MES
- 2-(N-morpholino)ethanesulfonic acid
- MICU
- mitochondrial calcium uptake protein
- MRAP
- MCU-regulating acidic patch
- M.W.
- molecular weight
- NMR
- nuclear magnetic resonance
- NTD
- N-terminal domain
- OD600
- optical density at 600 nm
- PMSF
- phenylmethylsulfonyl fluoride
- SAD
- single-wavelength anomalous diffraction
- SEC
- size exclusion chromatography
- SeMet
- selenomethionine
- SDS
- sodium dodecyl sulfate
- SSRF
- Shanghai Synchrotron Radiation Facility
- TM
- transmembrane