Functional roles of Mg ²⁺ binding sites in ion-dependent gating of a Mg ²⁺ channel, MgtE, revealed by solution NMR

Magnesium ions (Mg ²⁺ ) are divalent cations essential for various cellular functions. Mg ²⁺ homeostasis is maintained through Mg ²⁺ channels such as MgtE, a prokaryotic Mg ²⁺ channel whose gating is regulated by intracellular Mg ²⁺ levels. Our previous crystal structure of MgtE in the Mg ²⁺ -bound, closed state revealed the existence of seven crystallographically-independent Mg ²⁺ -binding sites, Mg1–Mg7. The role of Mg ²⁺ -binding to each site in channel closure remains unknown. Here, we investigated Mg ²⁺ -dependent changes in the structure and dynamics of MgtE using nuclear magnetic resonance spectroscopy. Mg ²⁺ -titration experiments, using wild-type and mutant forms of MgtE, revealed that the Mg ²⁺ binding sites Mg1, Mg2, Mg3, and Mg6, exhibited cooperativity and a higher affinity for Mg ²⁺ , enabling the remaining Mg ²⁺ binding sites, Mg4, Mg5, and Mg7, to play important roles in channel closure. This study revealed the role of each Mg ²⁺ -binding site in MgtE gating, underlying the mechanism of cellular Mg ²⁺ homeostasis.