MRS2 missense variation at Asp216 abrogates inhibitory Mg2+ binding, potentiating cell migration and apoptosis resistance.

Mitochondrial magnesium (Mg2+) is a crucial modulator of protein stability, enzymatic activity, ATP synthesis, and cell death. Mitochondrial RNA splicing protein 2 (MRS2) is the main Mg2+ channel in the inner mitochondrial membrane that mediates influx into the matrix. Recent cryo-electron microscopy (cryo-EM) human MRS2 structures exhibit minimal conformational changes at high and low Mg2+, yet the regulation of human MRS2 and orthologues by Mg2+ binding to analogous matrix domains has been well established. Further, a missense variation at D216 has been identified associated with malignant melanoma and MRS2 expression and activity is implicated in gastric cancer. Thus, to gain more mechanistic and functional insight into Mg2+ sensing by the human MRS2 matrix domain and the association with proliferative disease, we assessed the structural, biophysical, and functional effects of a D216Q mutant. We show that the D216Q mutation is sufficient to abrogate Mg2+-binding and associated conformational changes including increased α-helicity, stability, and monomerization. Further, we reveal that the MRS2 matrix domains interact with ~µM affinity, which is weakened by up to two orders of magnitude in the presence of Mg2+ for wild-type but unaffected for D216Q. Finally, we demonstrate the importance of Mg2+ sensing by MRS2 to prevent matrix Mg2+ overload as HeLa cells overexpressing MRS2 show enhanced Mg2+ uptake, cell migration, and resistance to apoptosis while MRS2 D216Q robustly potentiates these cancer phenotypes. Collectively, our findings further define the MRS2 matrix domain as a critical Mg2+ sensor that undergoes conformational and assembly changes upon Mg2+ interactions dependent on D216 to temper matrix Mg2+ overload.

The human MRS2 magnesium-binding domain is a regulatory feedback switch for channel activity.

Mitochondrial RNA splicing 2 (MRS2) forms a magnesium (Mg2+) entry protein channel in mitochondria. Whereas MRS2 contains two transmembrane domains constituting a pore on the inner mitochondrial membrane, most of the protein resides within the matrix. Yet, the precise structural and functional role of this obtrusive amino terminal domain (NTD) in human MRS2 is unknown. Here, we show that the MRS2 NTD self-associates into a homodimer, contrasting the pentameric assembly of CorA, an orthologous bacterial channel. Mg2+ and calcium suppress lower and higher order oligomerization of MRS2 NTD, whereas cobalt has no effect on the NTD but disassembles full-length MRS2. Mutating-pinpointed residues-mediating Mg2+ binding to the NTD not only selectively decreases Mg2+-binding affinity ∼sevenfold but also abrogates Mg2+ binding-induced secondary, tertiary, and quaternary structure changes. Disruption of NTD Mg2+ binding strikingly potentiates mitochondrial Mg2+ uptake in WT and Mrs2 knockout cells. Our work exposes a mechanism for human MRS2 autoregulation by negative feedback from the NTD and identifies a novel gain of function mutant with broad applicability to future Mg2+ signaling research.

Limiting Mrs2-dependent mitochondrial Mg2+ uptake induces metabolic programming in prolonged dietary stress.

The most abundant cellular divalent cations, Mg2+ (mM) and Ca2+ (nM-µM), antagonistically regulate divergent metabolic pathways with several orders of magnitude affinity preference, but the physiological significance of this competition remains elusive. In mice consuming a Western diet, genetic ablation of the mitochondrial Mg2+ channel Mrs2 prevents weight gain, enhances mitochondrial activity, decreases fat accumulation in the liver, and causes prominent browning of white adipose. Mrs2 deficiency restrains citrate efflux from the mitochondria, making it unavailable to support de novo lipogenesis. As citrate is an endogenous Mg2+ chelator, this may represent an adaptive response to a perceived deficit of the cation. Transcriptional profiling of liver and white adipose reveals higher expression of genes involved in glycolysis, ß-oxidation, thermogenesis, and HIF-1α-targets, in Mrs2-/- mice that are further enhanced under Western-diet-associated metabolic stress. Thus, lowering mMg2+ promotes metabolism and dampens diet-induced obesity and metabolic syndrome.