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. To date, only MgtE from Thermus thermophilus (MgtE_TT) 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 (MgtE_BF) is unique since it lacks the entire Mg²⁺-sensing N-domain but has conserved structural motifs in the 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 experiments show that MgtE_BF binds Mg²⁺ and ATP, similar to MgtE_TT. Importantly, we show that, despite lacking the N-domain, MgtE_BF 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 the TM-domain of MgtE_BF 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 MgtE_BF is significantly changed in functionally pertinent membrane environment, emphasizing the importance of lipid-protein interactions in MgtE gating mechanisms.