Fig. 3: Two Mg²⁺-binding sites in the MRS2 soluble domain and their role in channel activity regulation.

a Two Mg²⁺-binding sites are observed in the MRS2 soluble domain between subunits. Insets refer to regions highlighted in (b, c). b Mg²⁺ in the soluble domain coordinated by negatively charged residues including E243, D247, and E138 from one subunit and E312 from the neighboring subunit in soluble binding site 1. In the soluble binding site 2, Mg²⁺ is coordinated by E261, E263, and E267 from one subunit and E290, E293, E297 from the neighboring subunit. Water molecules in soluble sites 1 and 2 are shown in red spheres. c Detailed coordination of Mg²⁺ ions in the soluble site 1 and 2. d Salt bridge formed between R116-E291 pair across subunits. Enlarged view of the R116-E291 is shown (lower panel). e Comparison of the Mg²⁺-binding sites in the soluble domain between HsMRS2 and TmCorA. Structure of TmCorA in closed state (PDB: 3JCF) in pink is overlaid with the MRS2 structure in blue. The Mg²⁺ ions in the soluble domain of TmCorA and MRS2 are represented as spheres in red and green, respectively. f Mg²⁺-auxotrophic growth complementation assay using MRS2 and its mutants. Serially diluted Mg²⁺-auxotrophic E. coli (BW25113 ΔmgtA, ΔcorA, ΔyhiD DE3) containing corresponding plasmids were spotted onto LB plates with and without MgSO₄, and grown at 30 °C.