Examining the Interplay Between MRS2 and Magnesium: Implications in Mitochondrial Function

Magnesium (Mg2+) is an essential and versatile ion that regulates countless cellular phenomena, from ATP synthesis to enzyme catalysis, protein and nucleic acid stabilization and ion transport. Not surprisingly, intracellular and subcellular Mg2+ is tightly regulated, and dysfunctional Mg2+ homeostasis is implicated in various diseases including cancer, where it influences metabolic reprogramming, apoptosis and multidrug resistance (MDR). Mitochondrial RNA Splicing Protein 2 (MRS2) forms an ion channel that mediates Mg2+ influx into mitochondrial matrix (MM) and has emerged as a key player in regulating intracellular Mg2+. Nevertheless, the precise molecular mechanisms underlying the regulation of human MRS2 and roles in cancer remain enigmatic. To begin elucidating the structural mechanisms underlying MRS2 regulation, we expressed, isolated and biophysically characterized the large, matrix-oriented amino terminal domain (NTD). Our data reveal that MRS2-NTD self-associates, which can be robustly disrupted by Mg2+. Further, we identified D216 and D220 as key residues within the NTD responsible for Mg2+ binding-induced changes in secondary, tertiary and quaternary structure. Functionally, the mutation-induced abrogation in Mg2+ binding to the NTD led to enhanced mitochondrial Mg2+ uptake in mammalian cells, uncovering an NTD-driven regulation mechanism. Next, we looked more specifically at the role of NTD-dependent feedback regulation in hallmarks of cancer, finding that Mg2+ binding to the inhibitory site decreases the self-association affinity by up to two orders of magnitude. Further, we demonstrate that Mg2+ binding to the NTD inhibitory site causes allosteric changes to the domain far from the Mg2+ binding site, and disruption of this Mg2+ sensitivity leads to enhanced Mg2+ uptake, cell migration and resistance to apoptosis. Finally, we isolated and characterized the matrix-oriented carboxyl-terminal domain (CTD), revealing Mg2+-insensitive higher order oligomerization necessary for the pentameric assembly of full-length MRS2. Further, deleting the CTD impaired Mg2+ transport, suppressed cell migration, enhanced doxorubicin-mediated apoptosis and suppressed metabolic activity. Collectively, the work establishes the NTD as a matrix Mg2+ sensor involved in the negative feedback autoregulation of the MRS2 channel and the CTD as a critical assembly domain that may be a useful therapeutic target in the context of gastric cancer and other diseases associated with dysregulated Mg2+ homeostasis.