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.
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