Spatial distribution of cytoplasmic domains of the Mg2+-transporter MgtE, in a solution lacking Mg2+, revealed by paramagnetic relaxation enhancement

MgtE is a prokaryotic Mg2+ transporter that controls cellular Mg2+ concentrations. We previously reported crystal structures of the cytoplasmic region of MgtE, consisting of 2 domains, that is, N and CBS, in the Mg2+-free and Mg2+-bound forms. The Mg2+-binding sites lay at the interface of the 2 domains, making the Mg2+-bound form compact and globular. In the Mg2+-free structure, however, the domains are far apart, and the Mg2+-binding sites are destroyed. Therefore, it is unclear how Mg2+-free MgtE changes its conformation to accommodate Mg2+ ions. Here, we used paramagnetic relaxation enhancement (PRE) to characterize the relative orientation of the N and CBS domains in the absence of Mg2+ in solution. When the residues on the surface of the CBS domain were labeled with nitroxide tags, significant PRE effects were observed for the residues in the N domain. No single structure satisfied the PRE profiles, suggesting that the N and CBS domains are not fixed in a particular orientation in solution. We then conducted ensemble simulated annealing calculations in order to obtain the atomic probability density and visualize the spatial distribution of the N domain in solution. The results indicate that the N domain tends to occupy the space near its position in the Mg2+-bound crystal structure, facilitating efficient capture of Mg2+ with increased intracellular Mg2+ concentration, which is necessary to close the gate.

► The orientation of the N and CBS domains of apo MgtE in solution was analyzed by PRE.
► These domains were suggested not fixed in a particular orientation in solution.
► Their spatial distribution was visualized as the atomic probability density.
► Structural mechanism for the efficient capture of Mg2+ was suggested.