MRS2 Missense Variation at Asp216 Abrogates Inhibitory Mg Binding, Potentiating Cell Migration and Apoptosis Resistance

Mitochondrial magnesium (Mg) is a crucial modulator of protein stability, enzymatic activity, ATP synthesis, and cell death. Mitochondrial RNA splicing protein 2 (MRS2) is the main Mg 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 Mg, yet the regulation of human MRS2 and orthologues by Mg 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 Mg 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 Mg-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 Mg for wild-type but unaffected for D216Q. Finally, we demonstrate the importance of Mg sensing by MRS2 to prevent matrix Mg overload as HeLa cells overexpressing MRS2 show enhanced Mg 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 Mg sensor that undergoes conformational and assembly changes upon Mg interactions dependent on D216 to temper matrix Mg overload.