Structures in multiple conformations reveal distinct transition metal and proton pathways in an Nramp transporter

Abstract

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...

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

“…LeuT-fold-common among secondary transporters (23)-and conformations consistent with an alternating-access model (18,19). Transmembrane segments (TMs) 1, 4, 5, 6, and 10 undergo the greatest displacement relative to each other and to the remaining six TMs, which function as a "scaffold" to support those movements (19,24).…”

“…Transmembrane segments (TMs) 1, 4, 5, 6, and 10 undergo the greatest displacement relative to each other and to the remaining six TMs, which function as a "scaffold" to support those movements (19,24). Furthermore, while metal transport requires bulk conformational change between outward-and inward-open states, proton uniport occurs through the outward-open-but not inward-open-state (19). Thus, metal ions and protons both transit the external aqueous vestibule to reach their conserved binding site-D56 for protons, and D56 along with N59, M230, and the A53 and A227 carbonyls for metals (19,25)-in the center of the protein, but take separate pathways from there to reach the cytoplasm (19).…”

“…DraNramp consists of 11 TMs, the first 10 of which adopt the canonical LeuT inverted-repeat of two pseudosymmetric five-TM units. We used our previously published alignment of 6878 Nramp sequences that include the canonical "DPGN" and "MPH" motifs (19) and determined the positions at which hydrophilic residues predominate (defined as Ser + Thr + Tyr + Asn + Gln +Asp + Glu + His + Lys + Arg > 80%) ( Fig. S1).…”

“…1). Thus, predominantly hydrophobic packing between TMs 1b, 3, 6a, 8, and 10 seals this external vestibule in the inward-open state (18,19). In contrast, a number of hydrophilic positions flank the conserved metal-binding site in the middle of the membrane, and the cytoplasmic half of the protein is rich in hydrophilic residues.…”

“…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.…”