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. 2023 Nov 8;14(1):7207.
doi: 10.1038/s41467-023-42599-3.

Cryo-EM structures of human magnesium channel MRS2 reveal gating and regulatory mechanisms

Louis Tung Faat Lai  1 , Jayashree Balaraman  1 , Fei Zhou  1 , Doreen Matthies  2
Affiliations

Cryo-EM structures of human magnesium channel MRS2 reveal gating and regulatory mechanisms

Louis Tung Faat Lai et al. Nat Commun. .

Abstract

Magnesium ions (Mg2+) play an essential role in cellular physiology. In mitochondria, protein and ATP synthesis and various metabolic pathways are directly regulated by Mg2+. MRS2, a magnesium channel located in the inner mitochondrial membrane, mediates the influx of Mg2+ into the mitochondrial matrix and regulates Mg2+ homeostasis. Knockdown of MRS2 in human cells leads to reduced uptake of Mg2+ into mitochondria and disruption of the mitochondrial metabolism. Despite the importance of MRS2, the Mg2+ translocation and regulation mechanisms of MRS2 are still unclear. Here, using cryo-EM we report the structures of human MRS2 in the presence and absence of Mg2+ at 2.8 Å and 3.3 Å, respectively. From the homo-pentameric structures, we identify R332 and M336 as major gating residues, which are then tested using mutagenesis and two cellular divalent ion uptake assays. A network of hydrogen bonds is found connecting the gating residue R332 to the soluble domain, potentially regulating the gate. Two Mg2+-binding sites are identified in the MRS2 soluble domain, distinct from the two sites previously reported in CorA, a homolog of MRS2 in prokaryotes. Altogether, this study provides the molecular basis for understanding the Mg2+ translocation and regulatory mechanisms of MRS2.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Structure of human MRS2 in the presence of Mg2+.
a The 2.8 Å cryo-EM density of MRS2 shown in side and bottom views. Density corresponding to each subunit is delineated by different colors. b, c Fitted structural model of MRS2 shown as pentameric complex (b) and as single subunit (c). Magnesium ions bound to MRS2 are represented as green spheres. d Topology of MRS2 showing α-helices as rods and β-strands as slightly darker arrows.
Fig. 2
Fig. 2. Mg2+-translocation pathway in MRS2.
a Sliced side view of MRS2 illustrating the translocation pathway of Mg2+. The electrostatic potential of the surface is colored (from negative in red to positive in blue). Insets refer to regions highlighted in (b). b Enlarged view of the GMN motif and pore region highlighting residues coordinating Mg2+ ions. Potential gating residues M336 and R332 are labeled in red. Only helices from subunits B, C, and D are shown for clearer illustration. c Pore radius plot of MRS2. d, e Network of hydrogen bonds connecting gating residue R332 and the loop between soluble α-helix 3–4 of an adjacent subunit. Enlarged view of the interaction is shown in (e). f Mg2+-auxotrophic growth complementation assay using the empty vector and TmCorA (controls), 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. g Ni2+-sensitivity assay of BL21-(DE3) expressing TmCorA (control), MRS2 and its mutants. Serially diluted E. coli containing corresponding plasmids were spotted onto LB plates with and without 1.8 mM NiCl2, and grown at 30 °C overnight.
Fig. 3
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.
Fig. 4
Fig. 4. Structure of MRS2 under EDTA condition.
a Cryo-EM density map of MRS2 in the presence of 1 mM EDTA at 3.3 Å. b Model of the MRS2-EDTA structure. c Comparison of MRS2-Mg2+ and MRS2-EDTA showing the loss of Mg2+ beneath the R332-ring.
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