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.