Unique structural features in an Nramp metal transporter impart substrate-specific proton cotransport and a kinetic bias to favor import

Abstract

Natural resistance-associated macrophage protein (Nramp) transporters enable uptake of essential transition metal micronutrients in numerous biological contexts. These proteins are believed to function as secondary transporters that harness the electrochemical energy of proton gradients by “coupling” proton and metal transport. Here we use the Deinococcus radiodurans (Dra) Nramp homologue, for which we have determined crystal structures in multiple conformations, to investigate mechanistic details of metal and proton transport. We untangle the proton-metal coupling behavior of DraNramp into two distinct phenomena: ΔpH stimulation of metal transport rates and metal stimulation of proton transport. Surprisingly, metal type influences substrate stoichiometry, leading to manganese-proton cotransport but cadmium uniport, while proton uniport also occurs. Additionally, a physiological negative membrane potential is required for high-affinity metal uptake. To begin to understand how Nramp’s structure imparts these properties, we target a conserved salt-bridge network that forms a proton-transport pathway from the metal-binding site to the cytosol. Mutations to this network diminish voltage and ΔpH dependence of metal transport rates, alter substrate selectivity, perturb or eliminate metal-stimulated proton transport, and erode the directional bias favoring outward-to-inward metal transport under physiological-like conditions. Thus, this unique salt-bridge network may help Nramp-family transporters maximize metal uptake and reduce deleterious back-transport of acquired metals. We provide a new mechanistic model for Nramp proton-metal cotransport and propose that functional advantages may arise from deviations from the traditional model of symport.

Transmembrane helix 6b links proton- and metal-release pathways to drive conformational change in an Nramp transition metal transporter

“…2C) were all deleterious to Co 2+ and Mn 2+ transport ( Fig. 2D-E), with the exception of M230 mutants that preserved high Co 2+ transport as seen previously (4,17,19). Serine and asparagine replacements for Q378 preserved greater activity than alanine or leucine, indicating the importance of a hydrophilic residue at the position.…”

“…S1 and S2). We then measured relative rates of in vivo Co 2+ transport (WT K M ≈ 1 mM (4)) via our established colorimetric assay (17) and Mn 2+ transport (WT K M ≈ 3 μM (4,19)) with a new assay in which metal uptake is monitored through the increase in fluorescence of intracellular GCaMP6f (Fig. S3).…”

“…This network includes at least seven potentially protonatable sidechains and leads from proton-and metal-binding D56 (19) through a tight corridor between TMs 3, 4, 8, and 9 to the cytoplasm ( Fig. 4A-B) to provide a route for proton transport (4,19). In contrast to the external and intracellular vestibules proposed as metal entrance and release pathways, the helices and residues within this polar network, with the exception of the cytoplasmic end of TM4, undergo little rearrangement between our outward-open, inward-occluded, and inward-open structures (18,19).…”

“…To maintain metal homeostasis, organisms must acquire metal ions in sufficient quantity from their environment, traffic them to their proper destination, and safely store or excrete any excess to avoid toxicity (2,3). Natural resistance-associated macrophage proteins (Nramps) are transporters that harness the electrochemical energy of proton gradients and the membrane potential to power the uptake of divalent transition metals (4)(5)(6)(7). Nramp homologs scavenge manganese in bacteria (8) and acquire and traffic manganese and iron in plants (9) and fungi (10).…”

“…We developed the Deinococcus radiodurans (Dra)Nramp homolog as a model system to understand the general mechanism of this family of transporters (4,(17)(18)(19). DraNramp is the sole highaffinity Mn 2+ uptake system (20,21) for a species that maintains an exceptionally high intracellular Mn 2+ concentration as a resistance mechanism to radiation damage (22), and thus it may be a particularly robust transporter given the likely demand for high expression and activity in vivo.…”