Spatial distribution of cytoplasmic domains of the Mg ²⁺- transporter MgtE, in a solution lacking Mg ²⁺, revealed by paramagnetic relaxation enhancement

MgtE is a prokaryotic Mg ²⁺ transporter that controls cellular Mg ²⁺ concentrations. We previously reported crystal structures of the cytoplasmic region of MgtE, consisting of 2 domains, that is, N and CBS, in the Mg ²⁺-free and Mg ²⁺-bound forms. The Mg ²⁺-binding sites lay at the interface of the 2 domains, making the Mg ²⁺-bound form compact and globular. In the Mg ²⁺-free structure, however, the domains are far apart, and the Mg ²⁺-binding sites are destroyed. Therefore, it is unclear how Mg ²⁺-free MgtE changes its conformation to accommodate Mg ²⁺ ions. Here, we used paramagnetic relaxation enhancement (PRE) to characterize the relative orientation of the N and CBS domains in the absence of Mg ²⁺ 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 Mg ²⁺-bound crystal structure, facilitating efficient capture of Mg ²⁺ with increased intracellular Mg ²⁺ concentration, which is necessary to close the gate.