Phosphorylation of the mitochondrial calcium uniporter beta subunit matrix domain locks a conformation that suppresses mitochondrial calcium flux.

Abstract

Mitochondrial calcium (Ca2+) uptake is a central regulator of metabolism, signal transduction and cell death pathways. The mitochondrial calcium uniporter complex (mtCU) mediates this flux through a tetrameric pore comprised of MCU and MCU beta (MCUb) subunits. The large, matrix-oriented amino (N)-terminal domains (NTDs) of both paralogs are critical regulatory hubs, responsive to both ion binding and post-translational modifications. Despite high sequence identity between the MCU and MCUb NTDs, we previously identified significant disparities in regulatory ion-binding mechanisms. Further, while phosphorylation of MCU-NTD at Ser92 by protein kinase C (PKC) is known to induce a conformational change that regulates mtCU flux, whether the conserved post-translational modification site on MCUb serves a similar or divergent regulatory role remains unexplored. Here, using a colorimetric phosphate release and phosphoprotein staining assays, solution NMR spectroscopy and LC-MS/MS, we reveal that PKCδ phosphorylates MCUb-NTD at Ser77. Moreover, an S77D phosphomimetic induced structural and molecular dynamics behaviour consistent with the phosphorylated state. Remarkably, the S77D substitution alone was sufficient to drive the MCUb-NTD into a conformation analogous to the Ca2+-bound state, in silico and in vitro, bypassing the requirement for ion binding. This phosphorylation-induced transition exhibited increased thermal stability but weakened hetero-oligomerization with MCU-NTD compared to wild-type. Functionally, MCUb-S77D reduced mitochondrial Ca2+ uptake in HEK293T and HeLa cells without altering colocalization with MCU. Collectively, our results show that Ser77 phosphorylation locks the MCUb-NTD into a Ca2+-insensitive conformation that mimics the Ca2+-bound state, potentiating the suppression of mitochondrial Ca2+ flux.