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.