The structure of MgtE in the absence of magnesium provides new insights into channel gating

Abstract

MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed-state, and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. There are no structures of the transmembrane (TM) domain for MgtE in Mg2+-free conditions, and the pore-opening mechanism has thus remained unclear. Here, we determined the cryo-electron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE TM domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

Ion selectivity mechanism of the MgtE channel for Mg2+ over Ca2+

“…Notably, there was a certain proportion of the dimer fraction even in the presence of 5 mM EDTA and water (control) ( Figure 2 ), indicating the flexibility of the TM domain in the absence of divalent cations. This is consistent with the previous results of the biochemical cross-linking of MgtE 19 as well as the patch clamp recording of MgtE, showing the structural equilibrium of MgtE between open and closed conformations under Mg 2+ -free conditions. 14 Furthermore, the previous 57 Co 2+ uptake assay with Escherichia coli cells expressing MgtE showed the half-maximal inhibitory concentration ( K 0.5 ) value in the sub-100 μM order, presumably corresponding to the affinity of the MgtE pore region for Mg 2+ ions, because of the coordination chemistry of Mg 2+ similar to that of Co 2+ .…”

“…The close inspection shows that the conformational changes originate from the selectivity filter ( Figure 1 C). We verified these structural changes by cross-linking experiments with the cysteine-substituted mutant of MgtE at Leu421 and Thr336, 19 where the intersubunit distances of Cα atoms between Leu421 and Thr336 were 5.2 and 14.0 Å in the presence and absence of Mg 2+ ions, respectively ( Figure 1 D).…”

“…To estimate the selectivity of the selectivity filter of MgtE in the TM domain for Mg 2+ over Ca 2+ , using the MgtE T336C/L421C mutant lacking the N domain (MgtE ΔN T336C/L421C) and Cu 2+ phenanthroline as a catalyst, we performed biochemical cross-linking experiments similar to our previous MgtE experiments 19 and tested Mg 2+ and Ca 2+ at a gradient of concentrations ( Figure 2 ). Although MgtE also possesses regulatory Mg 2+ binding sites in the cytoplasmic domain, deletion of the N domain is known to abolish the Mg 2+ -sensitivity of the cytoplasmic domain.…”

“…Biochemical cross-linking experiments were performed as described previously. 19 First, 4 μL of the MgtE ΔN domain double cysteine mutant T336C/L421C construct protein at 0.5 mg/mL was mixed with 0.5 μL of EDTA at a final concentration of 5 mM and with MgCl 2 or CaCl 2 at appropriate concentrations, incubated on ice for 30 min, and then mixed with 0.5 μL of 10 mM freshly prepared Cu 2+ bis-1,10-phenanthroline (with a molar ratio of 1:3) and allowed to react on ice for another 30 min. The control samples and other samples were analyzed by SDS–PAGE under reducing conditions and nonreducing conditions, respectively.…”

Structural and functional comparison of magnesium transporters throughout evolution

“…4 A), which is involved in the gating mechanism of the protein. Decreased intracellular Mg 2+ levels give flexibility to the N-lobe and the plug, ultimately resulting in opening of the pore [ 20 , 83 , 86 , 87 ]. The transmembrane spanning domains contain conserved D1 and D2 domains, defined by PX 6 GN and P(D/A)X 4 PX 6 D motifs, respectively.…”