Novel Mg 2+ binding sites in the cytoplasmic domain of the MgtE Mg 2+ channels revealed by X-ray crystal structures

Abstract

MgtE is a Mg ²⁺-selective channel regulated by the intracellular Mg ²⁺ concentration. MgtE family proteins are highly conserved in all domains of life and contribute to cellular Mg ²⁺ homeostasis. In humans, mutations in the SLC41 proteins, the eukaryotic counterparts of the bacterial MgtE, are known to be associated with various diseases. The first MgtE structure from a thermophilic bacterium, Thermus thermophilus, revealed that MgtE forms a homodimer consisting of transmembrane and cytoplasmic domains with a plug helix connecting the two and that the cytoplasmic domain possesses multiple Mg ²⁺ binding sites. Structural and electrophysiological analyses revealed that the dissociation of Mg ²⁺ ions from the cytoplasmic domain induces structural changes in the cytoplasmic domain, leading to channel opening. Thus, previous works showed the importance of MgtE cytoplasmic Mg ²⁺ binding sites. Nevertheless, due to the limited structural information on MgtE from different species, the conservation and diversity of the cytoplasmic Mg ²⁺ binding site in MgtE family proteins remain unclear. Here, we report crystal structures of the Mg ²⁺-bound MgtE cytoplasmic domains from two different bacterial species, Chryseobacterium hispalense and Clostridiales bacterium, and identify multiple Mg ²⁺ binding sites, including ones that were not observed in the previous MgtE structure. These structures reveal the conservation and diversity of the cytoplasmic Mg ²⁺ binding site in the MgtE family proteins.

Keywords: crystal structure; ion channels; magnesium; metal homeostasis; regulation.

Figure 1

Comparison of the amino acid sequence and Mg ²⁺ binding sites (A‒C) Thermus thermophilus MgtE (TtMgtE) structure (PDB: 2YZ9). The N domain, CBS domain, plug helix and TM domain of one subunit are colored blue, green, yellow and red, respectively. The neighboring subunit is colored gray. Mg 2+ ions are shown as purple spheres. The overall structure (A) and close-up views of the Mg 2+ binding sites (B,C) are shown in cartoon representations. (D) Sequence alignment of Chryseobacterium hispalense MgtE (ChMgtE) (Accession number: WP_029295268.1), Clostridiales bacterium MgtE (CbMgtE) (Accession number: HCW50656.1), TtMgtE (Accession number: WP_011228410.1), Staphylococcus aureus MgtE (SaMgtE) (Accession number: WP_050596825.1), Bacillus subtilis MgtE (BsMgtE) (Accession number: WP_128441581.1), and Vibrio cholerae MgtE (VcMgtE) (Accession number: HAS4393389.1). The alignment and figure were generated using Clustal Omega [18] and ESPript [19]. Green, orange and purple circles indicate the Mg 2+ binding sites of ChMgtE, CbMgtE and TtMgtE, respectively. Numbers in the circles denote the numbering of the Mg 2+ binding sites.

Figure 2

In vivo complementation assay Serially diluted cultures (10 –2, 10 –3, 10 –4, 10 –5, 10 –6, and 10 –7) of Mg 2+ auxotrophic E. coli strain (BW25113 ΔmgtA ΔcorA ΔyhiD DE3) transformants harboring expression plasmids (vector, TtMgtE, CbMgtE and ChMgtE, respectively) were spotted onto the assay LB plates: (A) LB (50 μg/mL kanamycin, 100 mM MgSO 4), (B) LB (50 μg/mL kanamycin).

Figure 3

Chryseobacterium hispalense MgtE cytosolic domain structure (A‒C) ChMgtE cytoplasmic domain structure. The coloring scheme is the same as in Figure 1. Water molecules are shown as red spheres. Hydrogen and ionic bonds are indicated as dotted lines. The overall structure (A) and the close-up view of the Mg 2+ binding sites (B,C) are shown in cartoon representations.

Figure 4

Clostridiales bacterium MgtE cytosolic domain structure (A‒D) CbMgtE cytoplasmic domain structure. The coloring scheme is the same as in Figure 1. Water molecules are shown as red spheres. Hydrogen and ionic bonds are indicated as dotted lines. The overall structure (A) and close-up views of the Mg 2+ binding sites (B‒D) are shown in cartoon representation.

Figure 5

Structural comparison Superposition of the ChMgtE (green) and CbMgtE (orange) cytoplasmic domain structures onto the TtMgtE structure (violet). The close-up views of the Mg 2+ binding sites are shown in cartoon representation (A‒D). Mg 2+ ions (Mg1-Mg7 from the TtMgtE structure, Mg8-Mg9 from the ChMgtE structure, and Mg10 from the CbMgtE structure) are shown as purple spheres.