Functional roles of Mg²⁺ binding sites in ion-dependent gating of a Mg²⁺ channel, MgtE, revealed by solution NMR.

Abstract

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