Nramp family transporters—expressed in organisms from bacteria to humans—enable uptake of essential divalent transition metals via an alternating-access mechanism that also involves proton transport. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp in multiple conformations to provide a thorough description of the Nramp transport cycle by identifying the key intramolecular rearrangements and changes to the metal coordination sphere. Strikingly, while metal transport requires cycling from outward- to inward-open states, efficient proton transport still occurs in outward-locked (but not inward-locked) DraNramp. We propose a model in which metal and proton enter the transporter via the same external pathway to the binding site, but follow separate routes to the cytoplasm, which could facilitate the co-transport of two cationic species. Our results illustrate the flexibility of the LeuT fold to support a broad range of substrate transport and conformational change mechanisms.

Natural resistance-associated macrophage protein (Nramp) family transporters catalyze uptake of essential divalent transition metals like iron and manganese. To discriminate against abundant competitors, the Nramp metal-binding site should favor softer transition metals, which interact either covalently or ionically with coordinating molecules, over hard calcium and magnesium, which interact mainly ionically. The metal-binding site contains an unusual, but conserved, methionine, and its sulfur coordinates transition metal substrates, suggesting a vital role in their transport. Using a bacterial Nramp model system, we show that, surprisingly, this conserved methionine is dispensable for transport of the physiological manganese substrate and similar divalents iron and cobalt, with several small amino acid replacements still enabling robust uptake. Moreover, the methionine sulfur's presence makes the toxic metal cadmium a preferred substrate. However, a methionine-to-alanine substitution enables transport of calcium and magnesium. Thus, the putative evolutionary pressure to maintain the Nramp metal-binding methionine likely exists because it-more effectively than any other amino acid-increases selectivity for low-abundance transition metal transport in the presence of high-abundance divalents like calcium and magnesium.transition metals | MntH | divalent metal transporter DMT1 | hard-soft acid-base theory | ion selectivity filters A ll organisms require transition metal ions as cofactors in proteins that perform a variety of essential cellular tasks. Through evolution, organisms have developed mechanisms to acquire, transport, and safely store essential metals such as manganese, iron, cobalt, and zinc. The natural resistance-associated macrophage protein (Nramp) family of metal transporters represents a common transition metal acquisition strategy conserved across all kingdoms of life (1). The first discovered mammalian Nramp (Nramp1) is expressed in phagosomal membranes and likely extracts essential metals to help kill engulfed pathogens (2, 3). Mammals use Nramp2, an essential gene also called DMT1, to absorb dietary iron into the enterocytes that line the small intestine (4) and to extract iron from transferrin-containing endosomes in all tissues. Bacteria express their own Nramp homologs, which they typically use to scavenge manganese and other first row divalent transition metals (5, 6). Last, most plants have several Nramp homologs that take up iron and manganese, the essential cofactor in photosystem II, from the soil or vacuolar stores (7,8).Nramps are generally thought to function as metal-proton symporters (1) and are able to bind and/or transport a wide range of divalent transition metal substrates, including the biologically useful metals Mn 2+ , Fe 2+ , Co 2+ , Ni 2+ , Cu 2+ , and Zn 2+ , as well as the toxic heavy metals Cd 2+ , Pb 2+ , and Hg 2+ (4, 9-13). Nramps do discriminate against the divalent alkaline earth metal ions Mg 2+ and Ca 2+ (9, 14), which are typically several orders of magnitude mor...

Summary The widely-conserved natural resistance associated macrophage protein (Nramp) family of divalent metal transporters enables manganese import in bacteria and dietary iron uptake in mammals. We determined the crystal structure of the Deinococcus radiodurans Nramp homolog (DraNramp) in an inward-facing apo state, including the complete transmembrane (TM) segment 1a—absent from a previous Nramp structure. Mapping our cysteine accessibility scanning results onto this structure, we identified the metal permeation pathway in the alternate outward-open conformation. We investigated the functional impact of two natural anemia-causing glycine-to-arginine mutations, which impaired transition metal transport in both human Nramp2 and DraNramp. The TM4 G153R mutation perturbs the closing of the outward metal permeation pathway and alters the selectivity of the conserved metal-binding site. In contrast, the TM1a G45R mutation prevents conformational change by sterically blocking the essential movement of that helix, thus locking the transporter in an inward-facing state.

13Nramp family transporters-expressed in organisms from bacteria to humans-enable uptake of 14 essential divalent transition metals via an alternating-access mechanism that includes proton co-15 transport. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp at 16 complementary stages of its transport cycle to provide a thorough description of the Nramp 17 transport cycle by identifying the key intramolecular rearrangements and changes to the metal 18 coordination sphere. Strikingly, while metal transport requires cycling from outward-to inward-19 open states, efficient proton transport still occurs in outward-locked (but not inward-locked) 20DraNramp. We propose a model in which metal and proton enter the transporter via the same 21 external pathway to the binding site, but follow separate routes to the cytoplasm, thus resolving 22 the electrostatic dilemma of using a cation co-substrate to drive a cation primary substrate. Our 23 results illustrate the flexibility of the LeuT fold to support a broad range of co-substrate coupling 24 and conformational change mechanisms. 25 Krishnamurthy and Gouaux, 2012;Malinauskaite et al., 2014;Ressl et al., 2009; Shimamura et 48 al., 2010; Weyand et al., 2008). 49Natural resistance-associated macrophage proteins (Nramps) are APC-superfamily transition 50 metal transporters that enable uptake of rare micronutrients such as Mn 2+ in plants and bacteria 51 and Fe 2+ in animals (Cellier, 2012;Courville et al., 2006;Nevo and Nelson, 2006). Nramps bind 52 and/or transport biologically essential divalent metals such as Mn 2+ , Fe 2+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ -53 and toxic metals like Cd 2+ , Pb 2+ , and Hg 2+ -but not the abundant alkaline earth metals Mg 2+ and 54 Ca 2+ (Bozzi et al., 2016a;Ehrnstorfer et al., 2014). Metal uptake by Nramps involves proton co-55 transport, and many homologs also display considerable proton uniport-proton transport in the 56 absence of added metal that suggests loose coupling between the co-substrates (Chen et al., 1999; 57 Gunshin et al., 1997;Mackenzie et al., 2006;Nelson et al., 2002;Xu et al., 2004). Nramps have 58 11 or 12 TMs, the first ten forming a LeuT fold, as seen in structures of three bacterial Nramp 59 homologs (Bozzi et al., 2016b;Ehrnstorfer et al., 2014;Ehrnstorfer et al., 2017), including our 60 model system Deinococcus radiodurans (Dra)Nramp (Bozzi et al., 2016b). Conserved aspartate, 61 asparagine, and methionine residues in TM1 and TM6 coordinate transition metal substrates as 62 observed in an inward-open state (Ehrnstorfer et al., 2014), while only a metal-free outward-open 63 state has been reported (Ehrnstorfer et al., 2017). 64 Here we provide the first complementary structures of the same Nramp homolog in multiple 65 conformations, including the first metal-bound outward-open Nramp structure, and a novel 66inward-occluded structure. These allow us to fully illustrate the transport cycle for DraNramp. We 67 also show that metal transport requires the expected alternating access bulk confor...

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