Molecular Mechanism of Nramp-Family Transition Metal Transport

Abstract

The Natural resistance-associated macrophage protein (Nramp) family of transition metal transporters enables uptake and trafficking of essential micronutrients that all organisms must acquire to survive. Two decades after Nramps were identified as proton-driven, voltage-dependent secondary transporters, multiple Nramp crystal structures have begun to illustrate the fine details of the transport process and provide a new framework for understanding a wealth of preexisting biochemical data. Here we review the relevant literature pertaining to Nramps' biological roles and especially their conserved molecular mechanism, including our updated understanding of conformational change, metal binding and transport, substrate selectivity, proton transport, proton-metal coupling, and voltage dependence. We ultimately describe how the Nramp family has adapted the LeuT fold common to many secondary transporters to provide selective transition-metal transport with a mechanism that deviates from the canonical model of symport.

Keywords: APC superfamily; iron homeostasis; manganese; proton-coupled transport; secondary transporter.

Figure 1.. Functions and Evolution of the Nramp family.

(a) Known roles for Nramps in mammalian iron metabolism. In the acidic environment of the small intestines (Left), Fe³⁺ is reduced to Fe²⁺ by the duodenal cytochrome DCYTB or dietary ascorbic acid, then transported by NRAMP2 across the apical membrane to the enterocyte’s cytosol, where it can be oxidized and stored in ferritin or exported to the bloodstream via ferroportin. In the acidified phagosomes of macrophages (Upper center), free Fe²⁺ released from dying red blood cells or engulfed pathogens is extracted by NRAMP1 (and perhaps NRAMP2) to the cytosol and subsequently exported through ferroportin. Exported Fe²⁺ is oxidized by hephaestin to Fe³⁺, which then tightly binds to transferrin for distribution throughout the body (Lower center). At a destination cell such as a red blood cell precursor (Right), transferrin is endocytosed into an acidic endosome to release Fe³⁺, which is then reduced to Fe²⁺ by STEAP3 and transported by NRAMP2 to the cytosol. Fe²⁺ is then transported by NRAMP2 across the outer mitochondrial membrane and through mitoferrin into the mitochondrial matrix, where it can be oxidized and incorporated into heme. (b) A phylogenetic tree of a sampling of Nramp homologs illustrates the Nramp family’s evolutionary divergence into several major clades for prokaryotes, fungi, plants, and animals. Sequences were aligned using Geneious version 9.1 (Biomatters) and the BLOSUM62 matrix. Model homologs discussed in the text and aligned in Figure 2 are in bold.

Figure 2.. Sequence alignment of Nramp model systems and other clade-representative homologs.

(a) The Nramp secondary structure displays the characteristic topology of the LeuT fold, with TMs 1–5 and 6–10 comprising the two pseudosymmetric inverted repeats that intertwine in the tertiary structure. * denotes the location of the metal-binding site. TMs 1, 5, 6, and 10 are gold; TMs 3, 4, 8, and 9 are blue; TMs 2, 7, and 11 are gray. The same color scheme is used in later structure and model figures. Some homologs, including most eukaryotic Nramps and EcoleNramp [140], have a 12^th TM helix. (b) Comparison of sequence conservation among Nramp model systems and other clade-representative homologs. The percent identity is plotted in the top right of the matrix, and similarity in the bottom left (calculated using the BLOSUM62 scale). (c) Aligned sequences of these model homologs. The numbering and secondary structure of DraNramp are included above the alignment. Key residues for metal binding (magenta asterisks), proton transport and/or voltage dependence (cyan asterisks; blue asterisks indicate additional salt bridge in some homologs), and disease-causing mutation positions discussed in the main text (green highlight) are marked. Sequences were aligned using PSI/TM-Coffee [207] followed by manual editing of the N- and C-termini and some of the gap regions, and the alignment formatted with ESPript [208]. Uniprot accession numbers for the aligned sequences are: DraNramp (Q9RTP8), EcoliNramp (P0A769), Clostridium acetobutylicum MntH (Q97TN5; included as a representative of bacterial clade B), EcoleNramp (E4KPW4), ScaNramp (A0A0S4MEX1), S. cerevisiae Smf1p (C7GUZ9), Arabidopsis NRAMP1 (Q9SAH8), human NRAMP1 (P49279), mouse NRAMP2 (P49282), rat NRAMP2 (O54902), human NRAMP2 (P49281).

Figure 3.. Nramp crystal structures capture multiple stages of the transport cycle.

(a) Cartoon depiction of metal ion transport cycle by Nramps, with gold and blue lobes representing the mobile and stationary parts of the protein, respectively. Cartoons are labeled with currently available crystal structures. (b) A schematic representation of the main conformational changes in DraNramp between the outward- and inward-open states, reorienting TMs 1, 5, 6, and 10 (yellow; as illustrated, all but TM6b reorient to some extent). Metal binding may trigger the toppling of TM10 as well as TM6a’s inward movement to close off the outward metal-permeation pathway, with the latter motion propagated through extracellular loop 5–6 to pull on TM5 and thus begin to open the inner gate. TM1a must then displace significantly to fully expose the metal-binding site to the cytosol. (c) Crystal structures of DraNramp in three distinct states (PDB IDs: 6BU5, 6C3I, 6D91), with the outward and inward vestibules shown in gray dot surface. The changes in orientation of TM1a and TM4-TM5 are highlighted with an arrow and black lines, respectively. (d) Structure of outward-open EcoleNramp (PDB ID: 5M87), highlighting it similarities to the analogous DraNramp structure above. (e) A cylinder representation of the occluded DraNramp (PDB ID: 6C3I) with helices labeled, for comparisons of the Nramp fold to panels (b), (d), and (f). (f) Structure of inward-open ScaNramp (PDB ID: 5M95), highlighting its similarities to the analogous DraNramp structure above. Note that TM1a was deleted in the crystallized construct. All structures in panels (c)–(f) were superimposed using only the blue-colored regions (TMs 3, 4, 8, 9). Manganese ions are shown as magenta spheres, and empty metal-binding sites denoted by magenta asterisks.

Figure 4.. Modes of proton transport by DraNramp.

Proton transport occurs through the outward-open state, perhaps requiring subtle rearrangements. In contrast, metal transport requires bulk conformational change to the inward-open state. Figures adapted from Bozzi et al. 2019 [136].

Figure 5.. Conserved Nramp binding site changes across conformational states.

(a,b) Structures of outward-open DraNramp (a; PDB ID: 6BU5) and inward-open ScaNramp (b; PDB ID: 5M95; DraNramp residue numbering in parentheses) include bound Mn²⁺ substrate. In the outward-open DraNramp, D56, N59, M230 and the A53 carbonyl, along with two water molecules, coordinate Mn²⁺, while in the inward-open ScaNramp D56, N59, M230, and the A227 carbonyl coordinate Mn²⁺ (DraNramp residue numbering). (c) Model for changes in the metal-binding site during the transport cycle, including a possible switch from four to six to four metal-binding residues from the outward-open state, through a hypothetical occluded conformation (gray) in which Q378 may coordinate the metal, to the inward-open state. A change in metal coordination could serve to preferentially stabilize the conformational transition state to lower the activation energy barrier to transport. The surrounding binding-site residues may stabilize the negatively charged D56 during the return to the outward-open state, while D56 protonation likely then orients neighboring N59 to optimally bind incoming metal. Model adapted from Bozzi et al. 2019 [136]. (d) Mapped on the outward-open DraNramp structure are the Cα positions of residues from the inner metal coordination sphere (green), the outer selectivity sphere (brown) or the salt-bridge network (cyan) that influence metal selectivity, as discussed in the text.

Figure 6. . Conserved hydrophilic network forms Nramp proton-transport pathway.

(a) Structures of outward-open DraNramp (left; PDB ID: 6BU5) and EcoleNramp (right; PDB ID 5M87; DraNramp numbering in parentheses) show conserved network of protonatable residues on TMs 1, 6, 3, and 9 leading from metal-binding site to the cytosol, which is a unique feature of the Nramp-clade of the LeuT-fold family. (b) Model for multistep proton transport in DraNramp based on predicted pK_a values and mutagenesis data. The proton enters through the extracellular vestibule to bind to D56, is transferred to D131, facilitated by H232 and E134, and exits to the cytosol through a narrow passageway between TMs 3, 4, 8, and 9 that includes multiple additional charged and hydrophilic residues. (c) Model for Nramp proton-metal cotransport. (Left) A proton enters the extracellular vestibule and binds to D56. (Middle) Incoming metal displaces the proton as it binds to D56, N59, M230, and the A53 carbonyl. The proton passes to D131, stabilized by H232 and E134 during the transfer. Bulk conformational change occurs as modeled in Figure 3(b), possibly adding TM10’s Q378 and TM6’s A227 carbonyl to the metal-coordination sphere to drive the rearrangements. (Right) The proton is ultimately released to the cytosol through the TM3-TM9 salt-bridge network, a structural feature which also imparts a voltage dependence to this process that limits metal transport at lower magnitude ΔΨ. Metal substrate is released into the wide intracellular vestibule and the empty transporter can return to the outward-open state to repeat the cycle. Figure adapted from Bozzi et al. 2019 [132].

Figure 7.. Kinetic model of Nramp metal and proton transport.

(a) Nramp transport cycle diagram illustrating possible binding/unbinding and transport events. While metal transport requires bulk conformational change, proton uniport occurs through the outward-open state. Metal elemental identity controls whether either M²⁺:H⁺ cotransport (with a stoichiometry that may vary depending on voltage and pH) or M²⁺ uniport occurs. (b) Simplified, hypothetical free energy diagrams for Nramp transport events in a typical physiological context of higher [H⁺] outside, higher [M²⁺] inside, and a moderate negative-inside ΔΨ. Proton cotransport may significantly reduce the barrier to M²⁺ transport, although for some metals the kinetic barrier to uniport appears to be lower than that for cotransport. Voltage affects both the magnitude of the energetic barriers for metal transport as well as the relative ΔG for transport, while ΔpH affects the energetic barrier and, depending on the extent of thermodynamic coupling between metal and proton transport, the relative ΔG. The proposed mechanism for Nramps favors metal forward-transport over back-transport, which implies asymmetric kinetic barriers (and thus likely additional stable intermediate states), which are not shown in this model. Figures adapted from Bozzi et al. 2019 [132].