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. 2012 Nov 13;109(46):18809-14.
doi: 10.1073/pnas.1209018109. Epub 2012 Oct 29.

Structural asymmetry in the magnesium channel CorA points to sequential allosteric regulation

Roland Pfoh  1 , Angela Li, Nilmadhab Chakrabarti, Jian Payandeh, Régis Pomès, Emil F Pai
Affiliations

Structural asymmetry in the magnesium channel CorA points to sequential allosteric regulation

Roland Pfoh et al. Proc Natl Acad Sci U S A. .

Abstract

Magnesium ions (Mg(2+)) are essential for life, but the mechanisms regulating their transport into and out of cells remain poorly understood. The CorA-Mrs2-Alr1 superfamily of Mg(2+) channels represents the most prevalent group of proteins enabling Mg(2+) ions to cross membranes. Thermotoga maritima CorA (TmCorA) is the only member of this protein family whose complete 3D fold is known. Here, we report the crystal structure of a mutant in the presence and absence of divalent ions and compare it with previous divalent ion-bound TmCorA structures. With Mg(2+) present, this structure shows binding of a hydrated Mg(2+) ion to the periplasmic Gly-Met-Asn (GMN) motif, revealing clues of ion selectivity in this unique channel family. In the absence of Mg(2+), TmCorA displays an unexpected asymmetric conformation caused by radial and lateral tilts of protomers that leads to bending of the central, pore-lining helix. Molecular dynamics simulations support these movements, including a bell-like deflection. Mass spectrometric analysis confirms that major proteolytic cleavage occurs within a region that is selectively exposed by such a bell-like bending motion. Our results point to a sequential allosteric model of regulation, where intracellular Mg(2+) binding locks TmCorA in a symmetric, transport-incompetent conformation and loss of intracellular Mg(2+) causes an asymmetric, potentially influx-competent conformation of the channel.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
CorA pentamer in the closed state. (A) Side view: cartoon representations of two adjacent protomers, protomer A (gray, full length) and B (green, cytosolic domain). Helix α7 of protomer A is colored dark gray, and adjacent α7 helices have been omitted for clarity. The three remaining protomers C/D/E are shown in surface representation (light orange). Mg2+ ions bound to the divalent cation sensor (DCS) are shown as magenta colored spheres. (B) Top-to-bottom view: The color code is the same as in A; protomers are shown full length in cartoon representation.
Fig. 2.
Fig. 2.
Mg2+ sites inside the pore as observed in the X-ray structure of TmCorA-ΔNcc crystallized with Mg2+ present. (A) Five α7 helices (A to E) forming the CorA pore. Orange spheres indicate Mg2+ ions. (B and C) Close-up view of Mg2+ coordination at the GMN motif. Chain A is represented as a tube; a larger tube diameter indicates a higher B value. (D) Mg2+ coordination at the pore-lining residue S284 viewed along the pore axis. All distances in C and D are given in angstroms.
Fig. 5.
Fig. 5.
Influence of Mg2+ concentration on trypsin digestion. (A) SDS/PAGE analysis with identified species and cleavage sites shown for each susceptible site. (B) Sequence of TmCorA with cleavage sites is indicated by red arrows. Structural elements are color-coded matching the protein model representation in A. (C) Close-up view of the cleavage sites at R202/K205 and K292. (D) Close-up view of the N-terminal cleavage sites K9 and K22.
Fig. 3.
Fig. 3.
(A) Effect of divalent ions on the CorA conformation. Representations on the left show the locked state of TmCorA-WT in the presence of Mg2+, and those on the right show TmCorA-ΔNcc crystallized in the presence of Cs+ instead of divalent ions. All motions were deduced from superpositions with the locked state. (B) Definition of motions: radial tilt (blue), lateral tilt (red), and z rotation (magenta). Only the five α7 helices are shown with these motions indicated for the orange helix. The bell bending (green) is related to an increased kink between α7a and α7b of a single helix.
Fig. 4.
Fig. 4.
Analysis of structural fluctuations from 700-ns MD simulations of TmCorA-WT with and without Mg2+ ions present. Both simulations include Na+ for electroneutrality. Graphs A–C show only two of five distances for clarity. For the full set of distances, see Fig. S4. (A) Distance between K347 (Nζ) and E201′ (Cδ). (B) Distance between K292 (Nζ) and E204′ (Cδ). In A and B, a distance close to 3 Å indicates a salt bridge. (C) Distance between D253 (Cγ) and D89′ (Cγ). A distance between 4 and 6 Å indicates the presence of a neutralizing cation. (D) Close-up view of the formation of salt bridges monitored in A and B. (E) Close-up view of distances m1 and m1′′′′ monitored in C. (F) Angle between V248, L280, and I310 (all Cα). The graph is color coded to match the protomer representation in G. (G) Snapshot of the CorA pentamer and membrane after 600 ns of simulation. The phosphorus atoms of the 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayer are shown as blue spheres. (H) Water count in the MM stretch (constricted hydrophobic pore area from M302 to M291).
Fig. 6.
Fig. 6.
Model of the allosteric regulation of Mg2+ influx. The regulating Mg2+ ion is shown in pink, and the orange arrow indicates the direction of transport. Black arrows indicate the movements of the two adjacent protomers (shown in gray and green) in response to the loss of the regulatory divalent ion. The model is based on the results of both X-ray structures and MD simulations.
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