Structural insights into the ion selectivity of the MgtE channel for Mg ²⁺ over Ca ²⁺

MgtE is a Mg ²⁺ -selective ion channel whose orthologs are widely distributed from prokaryotes to eukaryotes, including humans, and play an important role in the maintenance of cellular Mg ²⁺ homeostasis. Previous functional analyses showed that MgtE transports divalent cations with high selectivity for Mg ²⁺ over Ca ²⁺ . Whereas the high-resolution structure determination of the MgtE transmembrane (TM) domain in complex with Mg ²⁺ ions revealed a Mg ²⁺ recognition mechanism of MgtE, the previous Ca ²⁺ -bound structure of the MgtE TM domain was determined only at moderate resolution (3.2 Å resolution), which was insufficient to visualize the water molecules coordinated to Ca ²⁺ ions. Thus, the structural basis of the ion selectivity of MgtE for Mg ²⁺ over Ca ²⁺ has remained unclear. Here, we showed that the metal-binding site of the MgtE TM domain binds to Mg ²⁺ ∼500-fold more strongly than Ca ²⁺ . We then determined the crystal structure of the MgtE TM domain in complex with Ca ²⁺ ions at a higher resolution (2.5 Å resolution), allowing us to reveal hexahydrated Ca ²⁺ , which is similarly observed in the previously determined Mg ²⁺ -bound structure but with extended metal-oxygen bond lengths. Our structural, biochemical, and computational analyses provide mechanistic insights into the ion selectivity of MgtE for Mg ²⁺ over Ca ²⁺ .