The natural resistance-associated macrophage protein (Nramp) family encompasses transition metal and proton cotransporters that are present in many organisms from bacteria to humans. Recent structures of Deinococcus radiodurans Nramp (DraNramp) in multiple conformations revealed the intramolecular rearrangements required for alternating access of the metal-binding site to the external or cytosolic environment. Here, using recombinant proteins and metal transport and cysteine accessibility assays, we demonstrate that two parallel cytoplasm-accessible networks of conserved hydrophilic residues in DraNramp, one lining the wide intracellular vestibule for metal release and the other forming a narrow proton transport pathway, are essential for metal transport. We further show that mutagenic or posttranslational modifications of transmembrane helix (TM) 6b, which structurally links these two pathways, impede normal conformational cycling and metal transport. TM6b contains two highly conserved histidines, His232 and His237. We found that different mutagenic perturbations of His232, just below the metal-binding site along the proton exit route, differentially affect DraNramp's conformational state, suggesting that His232 serves as a pivot point for conformational changes. In contrast, any replacement of His237, lining the metal exit route, locked the transporter in a transport-inactive outward-closed state. We conclude that these two histidines, and TM6b more broadly, help trigger the bulk rearrangement of DraNramp to the inward-open state upon metal binding and facilitate return of the empty transporter to an outward-open state upon metal release.

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Transmembrane helix 6b links proton-and metal-release pathways to drive conformational change in an Nramp transition metal transporter ABSTRACTThe natural resistance-associated macrophage protein (Nramp) family encompasses transition metal and proton co-transporters found in organisms from bacteria to humans. Recent structures of Deinococcus radiodurans (Dra)Nramp in multiple conformations revealed the intramolecular rearrangements required for alternating access. Here we demonstrate that two parallel cytoplasm-accessible networks of conserved hydrophilic residues in DraNramp-one lining the wide intracellular vestibule for metal release, the other forming a narrow proton-transport pathway-are essential for metal transport. We further show that mutagenic or post-translational modifications of transmembrane helix (TM) 6b, which structurally links these two pathways, impedes normal conformational cycling and metal transport. TM6b contains two highly conserved histidines, H232 and H237. Different mutagenic perturbations for H232, just below the metalbinding site along the proton-exit route, differentially affect DraNramp's conformational state, suggesting H232 serves as a pivot point for conformational change. In contrast, any tested replacement for H237, lining the metal-exit route, locks the transporter in a transport-inactive outward-closed state. We conclude that these two