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PreprintCited by 30Ion channel regulation and functionATP Synthase and ATPases ResearchAdvanced Electron Microscopy Techniques and Applications

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

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Lai, Louis Tung Faat, Balaraman, Jayashree, Zhou, Fei, Matthies, Doreen·2023·Nature Communications
DOI 10.1101/2023.08.22.553867PMC PMC10632456

Preprint — published as 10.1038/s41467-023-42599-3 · Paper page

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Abstract

Magnesium ions (Mg 2+ ) play an essential role in cellular physiology. In mitochondria, protein and ATP synthesis and various metabolic pathways are directly regulated by Mg 2+ . MRS2, a magnesium channel located in the inner mitochondrial membrane, mediates the influx of Mg 2+ into the mitochondrial matrix and regulates Mg 2+ homeostasis. Knockdown of MRS2 in human cells leads to reduced uptake of Mg 2+ into mitochondria and disruption of the mitochondrial metabolism. Despite the importance of MRS2, the Mg 2+ translocation and regulation mechanisms of MRS2 are still unclear. Here, using cryo-EM we determined the structure of human MRS2 in the presence and absence of Mg 2+ at 2.8 Å and 3.3 Å, respectively. From the homo-pentameric structures, we identified R332 and M336 as major gating residues, which were then tested using mutagenesis and two cellular divalent ion uptake assays. A network of hydrogen bonds was found connecting the gating residue R332 to the soluble domain, potentially regulating the gate. Two Mg 2+ -binding sites were 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 Mg 2+ translocation and regulatory mechanisms of MRS2.

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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. 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.CC BY · PubMed Central
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. 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.CC BY · PubMed Central
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. 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.CC BY · PubMed Central
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.
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.CC BY · PubMed Central

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Metadata

Authors
Lai, L. T. F., Balaraman, J., Zhou, F., & Matthies, D.
Affiliations
National Institutes of Health (US)
Corresponding author
Matthies, Doreen
ORCID
Venue
Nature Communications
Publisher
openRxiv
Published
2023-08-23
Volume
14
Issue
1
Pages
7207-7207
ISSN
2041-1723 (ISSN-L)
DOI
10.1101/2023.08.22.553867
PMC
PMC10632456
OpenAlex
W4388500134
Cited by
30
References
1
Citations by year
2026: 9 · 2025: 8 · 2024: 13
FWCI
2.59
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • U.S. Department of Health and Human Services (ZIA HD008998)
  • National Institutes of Health
  • National Institute of Child Health and Human Development
  • Eunice Kennedy Shriver National Institute of Child Health and Human Development (ZIAHD008998)
  • NHLBI Division of Intramural Research
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ion channel regulation and function
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › ATP Synthase and ATPases Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Advanced Electron Microscopy Techniques and Applications
Keywords
MRS2, magnesium channel, magnesium homeostasis, channel gating, magnesium binding sites, inner mitochondrial membrane, hydrogen bond network

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