Crystal Structure and Conformational Change Mechanism of a Bacterial Nramp-Family Divalent Metal Transporter

Abstract

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 that 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.

Keywords: LeuT fold; MntH; Nramp; crystallography; cysteine accessibility; divalent metal transporter; microcytic anemia; natural resistance-associated macrophage protein; transition metals.

Figure 1

DraNramp structure in the inward-facing state shows a highly kinked TM1. (A) Cartoon representation of DraNramp with TM helices labeled; the bundle (TMs 1, 2, 6, and 7) is gold, scaffold (TMs 3, 4, 5, 8, 9, and 10) blue, and TM11 gray. Dashed loops are disordered in the structure. (B) DraNramp topology diagram, with helices as cylinders, and gray trapezoids highlighting the inverted structural repeats (TMs 1-5 and TMs 6-10). Intracellular loop mutations Patch 1 to 3 in the crystallized construct are indicated. (C) Superposition of DraNramp (blue and gold) and ScaNramp (4WGW; gray and Mn²⁺ magenta) indicates a similar overall fold. The main differences are the position of TM5 (red arrowhead) and the presence of TM1a in DraNramp. Grey spheres mark the ScaNramp EEK motif corresponding to Patch 2, disordered in DraNramp. (D) Final 2F_o-F_c electron density map at 0.8σ showing density for TM1a. DraNramp is represented as a Cα trace. (E) Comparison of TM1 kink angle of DraNramp (yellow) with published LeuT-fold structures (gray; LeuT 2A65, 3TT1, 3TT3, 5JAE; Mhp1 2JLN, 4D1B, 2×79; vSGLT 2XQ2, 3DH4; BetP 4LLH, 4AIN, 4DOJ, 4C7R). The scaffolds of the corresponding structures were superimposed and oriented as in (A). (F) Conformational states in a transport cycle, color-coded as in (G). (G) The TM1b helices were aligned for DraNramp and three distinct LeuT conformations, highlighting the common kink at the unwound substrate-binding region in the middle of TM1. The kink is even more pronounced in inward-open DraNramp than any LeuT structure. See also Figure S1.

Figure 2

Cysteine accessibility scanning reveals the outward metal permeation pathway that is sealed shut in our crystallization construct. (A) Internal slice of the inward-facing DraNramp structure, including solvent accessibility of a panel of cysteine mutants spanning TM1, 3, and 6 using NEM. Spheres show Cα positions of highly NEM-protected (gray), outward-accessible (also MTSET and MTSEA-modified; red), inward-accessible (also MTSEA- but not MTSET- modified; cyan), or only NEM-accessible (black) residues. Accessibility is assessed as >50% NEM-modification in at least two separate experiments. Many outward-accessible residues, including A61C, are buried in our inward-open structure, suggesting they line an aqueous passage to the metal-binding site (approximate location labeled *) in an alternate outward-open conformation. (B) DraNramp’s proposed conformational equilibrium, in which A61C is solvent-accessible in the outward-open state, but buried (and thus NEM-protected) in the inward-open conformation. (C) The patch mutants in the crystallized DraNramp construct, tested alone or in combinations, have varying effects on in vivo Co²⁺ transport. While the 25-residue N-terminal truncation and patch 3 (RR398-9HH) did not impair function, patch 1 (QK169-70HH) reduced transport and patch 2 (EEK251-3YYY) completely eliminated transport. (D) While the transport-competent patch 1 and patch 3 mutants retained A61C accessibility (patch 1 at a reduced level), the transport-dead patch 2 mutant eliminated A61C accessibility, suggesting it locks the protein in the inward-open state. All data are averages ± s.d. (n ≥ 3). For reference, WT and EV Co²⁺ uptake time course and WT A61C accessibility data are repeated in subsequent figures. See also Figure S2 and Table S1.

Figure 3

Glycine-to-arginine mutations impair transition metal transport in both human Nramp2 and DraNramp. (A, B) Sequence logos of the TM1a region (A) showing that G45 (G75 in human Nramp2) on TM1a is absolutely conserved in Nramps, and a TM4 segment (B) showing that G153 (G185 in human/mouse/rat Nramp2) is generally a small amino acid. Logos were generated from a HMMER alignment of 2691 sequences using DraNramp to search the UniprotRef database, with an E-value cut-off of 1 × 10⁻⁹. Dra = D. radiodurans MntH, Sca = S. capitis MntH, Hs2 = Homo sapiens Nramp2, Mm2 = Mus musculus Nramp2, Rn2 = Rattus norvegicus Nramp2. (C, D) Fura-2 fluorescence quenching traces showing severe loss of function (no transport activity above baseline) for both G-to-R mutants compared to WT human Nramp2 for transport of the transition metals Fe²⁺ (C) and Cd²⁺ (D) in transfected HEK cells. Traces are representative of at least three independent transfection experiments. (E) Relative Fe²⁺ uptake of E. coli expressing the analogous G-to-R DraNramp mutants also showed significantly decreased transport activity compared to WT. Plotted are averages ± s.d. (n = 3). (F) Both G-to-R DraNramp mutants had decreased Cd²⁺ transport when reconstituted into proteoliposomes. Traces are representative of three experiments. EV = empty vector/vesicle. See also Figure S3.

Figure 4

G153R mutation perturbs outward-facing state and alters metal selectivity of binding site to favor Ca²⁺. (A) In the inward-facing DraNramp structure, G153 is located on the extracellular end of TM4 tightly packed in the back of the scaffold, far from the conserved metal-binding site. The G153 Cα is a magenta sphere; nearby residues are shown as transparent spheres with sidechains as sticks. (B) Co²⁺ uptake data showing charged bulky substitutions at G153 reduced transport, while aromatic substitutions retained WT-level transport. (C) G153R showed increased accessibility to NEM modification of the A61C outward-open conformational reporter, and G153F showed a further increase. (D) In in vivo Fe²⁺ uptake, G153R showed little additional effect when combined with metal-binding site mutations, and its residual Fe²⁺ transport still uses the conserved metal-binding site as double mutations G153R/D56A and G153R/N59A both severely reduced transport. (E) Competing Ca²⁺ reduced in vivo Fe²⁺ uptake more for G153R than WT, and double mutant G153R/M230A was even more susceptible to Ca²⁺ competition. The Ca²⁺-free Fe²⁺ uptake level (2 min uptake for WT/M230A; 15 min uptake for G153 mutants) was set to 100% for each variant to facilitate direct % inhibition comparison. (F) G153R transported more Ca²⁺ (as detected by Fura-2) than WT or the G45R mutant in an in vitro proteoliposome assay. All data are averages ± s.d. (n ≥ 3). See also Figure S4.

Figure 5

G45R mutation inhibits transport by locking Nramp in outward-closed conformation. (A) G45 (green sphere) is on TM1a on the intracellular side of the bundle, 11 residues below the metal-binding D56. (B) G45R and G45F were significant impaired in in vivo Co²⁺ transport. (C) G45F also impaired in vivo Fe²⁺ transport (WT, G45R and EV data reproduced from Figure 3E for comparison). (D) G45R and G45F drastically decreased accessibility of outside-open conformational reporter A61C, indicating a strong preference for the inward-open state. (E) Western blot showing G45R and G45F expressed similarly to WT DraNramp. All data are averages ± s.d. (n ≥ 3).

Figure 6

Mutations of conserved glycines shift DraNramp’s conformational landscape. (A) Cysteine modification as a function of NEM concentration for five extracellularly-accessible reporters, mapped on a top view of the structure (left). * indicates the metal-binding site. A61C data are repeated from Figures 3C and 4C for comparison. (B) NEM modification for five intracellularly-accessible cysteine reporters as well as T130C, which could not clearly be assigned as intracellular or extracellularly accessible. Reporter positions are indicated on the structure, viewed down the cytoplasmic vestibule. All data are averages ± s.d. (n ≥ 4). See also Figure S5.

Figure 7

TM1a movement is essential to the conformational change that opens the outward metal permeation pathway, thus enabling Nramp metal transport. (A) Initial (6 min) in vivo Co²⁺ transport (minus EV control) for accessible single-cysteine mutants along TM1 that were either left unmodified (black bars) or pre-reacted with NEM (3 mM; green bars). NEM modification greatly impaired transport for six out of the seven TM1a positions where cysteine mutants had high transport activity. Data are averages ± s.d. (n = 3). Western blots show all introduced cysteines were efficiently NEM-labeled, as preincubation with NEM (+) prevented formation of PEG5K-maleimide DraNramp [upper band in the (−) lanes]. R211C on extracellular loop 5-6 was readily NEM-modified without affecting activity. Endogenous C382 on TM10 in WT was not modified by NEM, and thus was fully modified by PEG5K-maleimide. The cysteine-less C382S is not labeled by either NEM or 5KPEG-maleimide. (B) Model of the conformational change process in DraNramp. Our metal transport and cysteine accessibility results demonstrated that the unencumbered TM1a movement is essential for conformational change into the outward-facing state, including evicting loop 7-8 which caps the outside metal permeation pathway and opening the interface between the bundle (TM1b and 6a) and scaffold (TM3, 8, and 10), thus allowing periplasmic metal ions to reach the binding site in the unwound regions of TM1 and 6 at the center of the membrane plane. See also Figure S6.