Characterization of a novel MgtE homolog and its structural dynamics in membrane-mimetics.

Magnesium (Mg2+ ) is the most abundant divalent cation in the cell and is critical for numerous cellular processes. Despite its importance, the mechanisms of intracellular Mg2+ transport and its regulation are poorly understood. MgtE is the main Mg2+ transport system in almost half of bacterial species, and is an ortholog of mammalian SLC41A1 transporters, which are implicated in neurodegenerative diseases and cancer. Till date, only MgtE from Thermus thermophilus (MgtETT ) has been extensively characterized mostly in detergent micelles, and gating-related structural dynamics in biologically-relevant membranes are scarce. The MgtE homolog from Bacillus firmus (MgtEBF ) is unique since it lacks the entire Mg2+ -sensing N-domain, but has conserved structural motifs in TM-domain for Mg2+ transport. In this work, we have successfully purified this novel homolog in a stable and functional form, and ColabFold structure prediction analysis suggests a homodimer. Further, microscale thermophoresis (MST) experiments show that MgtEBF binds Mg2+ and ATP, similar to MgtETT . Importantly, we show that, despite lacking the N-domain, MgtEBF mediates Mg2+ transport function in the presence of an inwardly directed Mg2+ gradient in reconstituted proteoliposomes. Furthermore, comparison of the organization and dynamics of Trp residues in TM-domain of MgtEBF in membrane-mimetics, in apo- and Mg2+ -bound forms, suggests that the cytoplasmic binding of Mg2+ might involve modest gating-related conformational changes at the TM-domain. Overall, our results show that the gating-related structural dynamics (hydration dynamics, conformational heterogeneity) of the full-length MgtEBF is significantly changed in functionally-pertinent membrane environment, emphasizing the importance of lipid-protein interactions in MgtE gating mechanisms.