Structural And Mechanistic Insights Into MICU1Regulation Of Mitochondrial Calcium Uptake

Mitochondria are the power plants of the cell and function as important nodes within signaling pathways to regulate various cellular activities. Calcium in the mitochondria plays a key role in several important functions, such as controlling the metabolic rate for cellular ATP production, the modulation of the amplitude and shape of the cytosolic Ca2+transients, and the induction of apoptosis through the release of cytochrome c from the mitochondrial intermembrane space into the cytosolic space.Mitochondrial Ca2+uptake was discovered in the1960s and was found to be mediated by the mitochondrial Ca2+uniporter (MCU). In2010, based on clues from comparative physiology, evolutionary genomics, organelle proteomics, and genome-wide RNAi screening, the mitochondrial calcium uptake1(MICU1) protein was identified as an essential element of mitochondrial calcium uptake.MICU1is a54kDa protein which can interact with MCU. It contains two EF-hands and can bind Ca2+. MICU1serves as a Ca2+sensor that regulates MCU. Recent study suggests that MICU1has two distinct functions in the regulation of mitochondrial calcium uptake:(1) it sets a [Ca2+]c threshold to inhibit Ca2+transport into mitochondria;(2) it provides cooperative activation as [Ca2+]c rises.Although we have some understanding of the functions of MICU1, its molecular mechanisms of calcium sense and MCU regulation remains unclear. Our study described the atomic structures of both Ca2+-free and Ca2+-bound human MICU1. Then we clarify the mechanism of MICU1sensing calcium and regulating mitochondrial calcium uptake based on structural analysis in combination with biochemistry and cell biology functional research.Our study suggest that Ca2+-free MICU1-xtal forms a hexamer which can binds and inhibits MCU. Upon Ca2+binding, MICU1undergoes large conformational changes, resulting in formation of multiple oligomers to activate MCU.In the hexamer of Ca2+-free MICU1-xtal, we find that the unusual C-helix plays a key role in MICU1oligomerization. Co-immunoprecipitation results suggests that C-helix can mediate the interaction of MICU1and MCU. The lack of C-helix can result in the complete lost of MICU1activated mitochondrial calcium uptake.In the structures, both Ca2+-free MICU1-xtal-deltaC and Ca2+-bound MICU1-xtal-deltaC are dimer, but they have different packing pattern. Destroy the two pairs of MICU1dimer can partly damage MICU1activated mitochondrial calcium uptake.We also demonstrate that the affinity of MICU1for Ca2+is approximately15-20μM, which indicated that MICU1is less likely to bind Ca2+in the resting cell.In conclusion, our work describes the crystal structures of Ca2+-free and Ca2+-bound human MICU1, which offer first and significant structural insights into the mitochondrial calcium transport machinery.