Bacteria require transition metals like manganese (Mn) for proliferation and pathogenesis, yet mismanagement of these essential nutrients can induce fatal metal intoxication or starvation. To maintain this precise and dynamic homeostasis, bacteria evolved systems of metalloregulators and integral membrane transporters, and these proteins are implicated in bacterial virulence upon invasion of mammalian hosts. MneP is the primary Mn(II) exporter in Bacillus subtilis and a member of the ubiquitous cation diffusion facilitator (CDF) family, yet its structure and mechanism remain unsolved. Building on our progress presented last year, we are working toward structurally characterizing MneP using cryogenic electron microscopy. To facilitate particle picking and alignment, we present an improved chimeric construct that fuses a small, antibody fragment-binding soluble protein to MneP via a rigid helical linker. In tandem, we developed a fluorescence-based transport assay to establish MneP’s functional capabilities and probe the impact of mutagenesis to its putative primary and secondary Mn-binding sites along with its charge interlock. The mechanistic insights garnered from coupling these structural and functional data will not only shed light on the workings of CDFs but also open new avenues for developing innovative therapeutics to inhibit deadly bacterial pathogens.
Boutet et al. (Sun,) studied this question.