线粒体镁 (Mg2) 是蛋白质稳定性、酶活性、ATP 合成和细胞死亡的重要调节剂。线粒体 RNA 剪接蛋白 2 (MRS2) 是线粒体内膜中主要的 Mg2 通道，介导流入基质。最近的冷冻电子显微镜 (cryo-EM) 人类 MRS2 结构在高和低 Mg2 条件下表现出最小的构象变化，但 Mg2 与类似基质结构域结合对人类 MRS2 和直系同源物的调节已经得到很好的确立。此外，D216 的错义变异已被鉴定与恶性黑色素瘤相关，并且 MRS2 的表达和活性与胃癌有关。因此，为了更深入地了解人类 MRS2 基质域对 Mg2 的感应以及与增殖性疾病的关联，我们评估了 D216Q 突变体的结构、生物物理和功能效应。我们表明，D216Q 突变足以消除 Mg2 结合和相关的构象变化，包括增加的 α 螺旋性、稳定性和单体化。此外，我们揭示了 MRS2 基质结构域与 ~μM 亲和力相互作用，在 Mg2 存在的情况下，野生型的亲和力减弱了两个数量级，但 D216Q 不受影响。最后，我们证明了 MRS2 的 Mg2 感应对于防止基质 Mg2 过载的重要性，因为过表达 MRS2 的 HeLa 细胞显示出增强的 Mg2 摄取、细胞迁移和抗凋亡能力，而 MRS2 D216Q 则显着增强这些癌症表型。总的来说，我们的研究结果进一步将 MRS2 矩阵域定义为一种关键的 Mg2 传感器，它会根据依赖于 D216 的 Mg2 相互作用而发生构象和组装变化，以调节矩阵 Mg2 过载。© 2024 作者。 《蛋白质科学》由 Wiley periodicals LLC 代表蛋白质协会出版。

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.© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.