Vibrational and Molecular Properties of Mg²⁺ Binding and Ion Selectivity in the Magnesium Channel MgtE

Magnesium ions (Mg²⁺) are crucial for various biological processes. A bacterial Mg²⁺ channel, MgtE, tightly regulates the intracellular Mg²⁺ concentration. Previous X-ray crystal structures showed that MgtE forms a dimeric structure composed of a total of 10 transmembrane α helices forming a central pore, and intracellular soluble domains constituting a Mg²⁺ sensor. The ion selectivity for Mg²⁺ over Ca²⁺ resides at a central cavity in the transmembrane pore of MgtE, involving a conserved aspartate residue (Asp432) from each monomer. Here, we applied ion-exchange-induced difference FTIR spectroscopy to analyze the interactions between MgtE and divalent cations, Mg²⁺ and Ca²⁺. Using site-directed mutagenesis, vibrational bands at 1421 (Mg²⁺), 1407 (Mg²⁺), ∼1440 (Ca²⁺), and 1390 (Ca²⁺) cm^-1 were assigned to symmetric carboxylate stretching modes of Asp432, involved in the ion coordination. Conservative modifications of the central cavity by Asp432Glu or Ala417Leu mutations resulted in the disappearance of the Mg²⁺-sensitive carboxylate bands, suggesting a highly optimized geometry for accommodating a Mg²⁺ ion. The dependency of the vibrational changes on Mg²⁺ and Ca²⁺ concentrations revealed the presence of a two different classes of binding sites: a high affinity site for Mg²⁺ ( K_d ≈ 0.3 mM) with low Ca²⁺ affinity ( K_d ≈ 80 mM), and a medium affinity site for Mg²⁺ ( K_d ≈ 2 mM) and Ca²⁺ ( K_d ≈ 6 mM), tentatively assigned to the central cavity and the sensor domain, respectively. With the aid of molecular dynamics simulation and normal-mode analysis by quantum chemistry, we confirm that changes in carboxylate bands of the high affinity binding site originate from Asp432 in the central cavity.