
==== Front
J Biol Chem
J Biol Chem
The Journal of Biological Chemistry
0021-9258
1083-351X
American Society for Biochemistry and Molecular Biology

S0021-9258(24)02090-8
10.1016/j.jbc.2024.107589
107589
JBC Reviews
In vitro reconstitution of transition metal transporters
Ongey Elvis L.
Banerjee Anirban anirban.banerjee@nih.gov
∗
Cell Biology and Neurobiology Branch, National Institutes of Child Health and Human, Development, National Institutes of Health, Bethesda, Maryland, USA
∗ For correspondence: Anirban Banerjee anirban.banerjee@nih.gov
19 7 2024
8 2024
19 7 2024
300 8 10758924 2 2024
28 6 2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Transition metal ions are critically important across all kingdoms of life. The chemical properties of iron, copper, zinc, manganese, cobalt, and nickel make them very attractive for use as cofactors in metalloenzymes and/or metalloproteins. Their versatile chemistry in aqueous solution enables them to function both as electron donors and acceptors, and thus participate in both reduction and oxidation reactions respectively. Transition metal ions can also function as nonredox multidentate coordination sites that play essential roles in macromolecular structure and function. Malfunction in transition metal transport and homeostasis has been linked to a wide number of human diseases including cancer, diabetes, and neurodegenerative disorders. Transition metal transporters are central players in the physiology of transition metals whereby they move transition metals in and out of cellular compartments. In this review, we provide a comprehensive overview of in vitro reconstitution of the activity of integral membrane transition metal transporters and discuss strategies that have been successfully implemented to overcome the challenges. We also discuss recent advances in our understanding of transition metal transport mechanisms and the techniques that are currently used to decipher the molecular basis of transport activities of these proteins. Deep mechanistic insights into transition metal transport systems will be essential to understand their malfunction in human diseases and target them for potential therapeutic strategies.

Keywords

transition metal
transition metal transport
metal transporter
membrane transport
In vitro reconstitution
proteoliposome
transport assay
membrane protein
iron transport
zinc transport
fluorescence
liposome
Abbreviations

DMT1 divalent metal-ion transporter-1

FPN ferroportin

ITC isothermal titration calorimetry

ZIP Zrt- and Irt-like protein

Reviewed by members of the JBC Editorial Board. Edited by Mike Shipston
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pmcMetal ions are among the essential chemical elements that all life forms must obtain from environmental sources and maintain within physiological limits to ensure survival, growth, and reproduction. Genetic disorder or malnutrition can lead to elevated levels or shortages of these vital life elements, causing severe diseases or even death. Transition metal ions occupy a special position in biology by virtue of their ability to form multidentate interactions and/or participate in redox chemistry. They function as cofactors in enzymes and/or metalloproteins which participate in a wide range of essential physiological processes. Biologically important transition metal ions are typically iron (Fe2+/3+), copper (Cu2+), zinc (Zn2+), manganese (Mn2+), cobalt (Co2+), nickel (Ni2+), and molybdenum (Mo2+). Transition metal cofactors can either play a structural role or can actively participate in reaction chemistry, thereby tremendously extending the repertoire of the chemistries accessible to biological macromolecules (1).

Of all the transition metals, iron is by far the most widely used in biological systems, primarily due to its highly versatile chemistry in aqueous solution where it can readily function both as an electron donor and an acceptor, and hence can facilitate both reduction and oxidation reactions respectively (2, 3). In eukaryotes, mitochondria play a centrally important role in the cell biology of iron. A critical component of heme biosynthesis, viz. the insertion of iron into globin to form hemoglobin, occurs within the organelle. Mitochondria is also the site for the synthesis of iron-sulfur clusters, that together with iron-containing cytochromes, form the backbone of the electron transport chain that results in synthesis of ATP (4). Other transition metal ions such as Cu2+ and Mn2+ also participate in redox pathways within biological systems, whereas redox-inert metal ions such as Zn2+ fulfill structural roles in proteins by stabilizing negative charges and/or serving in the active sites of enzymes by virtue of their intrinsic Lewis acid properties (5).

Unlike organic molecules such as lipids and sugars that can be broken down by enzymes to generate energy and biomass needed by cells, metals pose significant challenges to biological systems. For instance, environmental transition metals are mostly available in oxidized forms that generally exhibit poor solubility in water, and hence, are largely unsuitable for biological assimilation. The scarcity of bioavailable metal ion pools creates a thermodynamic challenge for organisms that need to concentrate these nutrients in cellular and subcellular compartments. To overcome these barriers, nature has evolved specific mechanisms involving myriad transport machineries and accessory proteins or chaperones (6). Transmembrane transporters are key players involved in maintaining the physiological concentrations of these metal ions, owing to their ability to harness preexisting electrochemical gradients to drive the transport of a desired substrate across the lipid bilayer (1).

The detailed chemical mechanisms of transition metal transport and homeostasis are relatively understudied and thus, poorly understood. The divalent metal-ion transporter-1 (DMT1) was the first mammalian transmembrane iron transporter to be identified more than a quarter of a century ago (7, 8, 9). Although domain structure and mutational analyses have lent useful insights into the structure-function relationships of DMT1 (10, 11), there is no report of in vitro reconstitution of the metal ion transport activity of purified full-length DMT1. This is in part because DMT1 is a eukaryotic transmembrane protein and consequently, challenging to isolate and purify. Another challenge with transition metal transporters is that attempts at overexpression during the course of purification may also promote cell death due to metal toxicity (12, 13, 14). Given the double-edged sword-like nature of transition metal ions, cells can only tolerate a defined level of trace metals to maintain viability. Below this threshold limit, the functions of critical metal-binding enzymes are compromised, and excess can overwhelm a cell, causing cell death (15). Although, overexpression, detergent extraction and purification of active membrane transporters are nontrivial tasks, the biochemical properties of a few transition metal transporters have been characterized in vitro, and three-dimensional structures of a few of them have been reported stemming from recent advances in the structural biology of membrane proteins (see Table 1).Table 1 Source, physiological substrate(s), and transport mechanisms of selected transition metal transporters

Protein name	Native source	In vitro data	Physiological substrate	Transport mechanism	Reference	
ZitB	Escherichia coli	Biochemical characterization	Zn2+	Zn2+/H+ antiport	(19, 34)	
Yiip	E. coli	Crystal structure	Zn2+	Zn2+/H+ antiport	(35, 36, 37, 38)	
BbZIP	Bordetella bronchiseptica	Crystal Structure	Zn2+	unknown	(39, 40)	
ScaNramp	Staphylococcus capitis	Crystal structure	Mn2+	Mn2+/H+ symport	(41)	
DraNramp	Deinococcus radiodurans	Crystal structure	Mn2+	• Mn2+/H+ symport

• Cd2+ uniport

• H+ uniport

	(28, 42)	
EcoNramp	Eremococcus coleocola	Crystal structure	Mn2+	Mn2+/H+ uniport	(10, 43)	
Nramp2/DMT1/DCT1	Homo sapiens	Biochemical characterization	Fe2+	Proton-coupled voltage-dependent symport	(44, 45)	
TMfrn1	Oreochromis niloticus	Biochemical characterization	Fe2+	Fe2+ uniporta	(46)	
FPN	Homo sapiens	Cryo-EM	Fe2+	Fe2+/H+ symport	(47, 48)	
BbFPN	Bdellovibrio bacteriovorus	Crystal structure	Fe2+	Fe2+ uniport	(49)	
TsFPN	Tarcius syrichta	Crystal structure	Fe2+	Fe2+/H+ antiport	(50)	
a remains to be determined if the symport mechanism is thermodynamically coupled.

Despite the importance of transition metal transport in biology, very few transition metal transporters have been studied by in vitro biochemical reconstitution in proteoliposomes with purified protein. Redox active transport metal ions can oxidize lipids, making them challenging transport substrates and thus in vitro reconstitution has lagged behind even structural studies, although the latter is considered to be the most important roadblock for understanding transmembrane protein mechanism. In this review, we focus on four groups of transition metal transporters for zinc, manganese, iron, and copper where in vitro reconstitution has been successfully used to investigate their transport mechanisms. Because of the specific aim of our review, we only highlight transition metal transporters that have been studied by in vitro reconstitution methods. In the context of the transporters discussed here, we also touch upon the inherent challenges of isolating and purifying the protein. Finally, we also summarize current knowledge on the use of transition metal transporters as target for therapeutic development.

Reconstitution techniques to investigate membrane protein transport function

Experiments to investigate transition metal transporters may be empirically grouped into three main categories, namely, in vivo, in vitro, and in cellulo. We use in cellulo to describe experiments conducted in cell cultures and distinguish them from in vitro experiments that are conducted in test tubes using purified or extracted components. Early characterization of transition metal transporters including substrate identification and mode of transport was mostly achieved through in vivo and in cellulo assays and through genetic manipulation and observing the resulting phenotypes (7, 8, 16, 17, 18, 19). The obvious benefit of such techniques is that the experiments are conducted in the native environment with all the physiologically relevant components in place. However, interpretations of the results are usually complicated due to the possible participation of these components, which cannot be controlled easily. For example, intracellular pH is difficult to control and metal efflux from cells is difficult to quantify since chaperone proteins or small chelator molecules can sequester metal ions injected into the cells (20). Therefore, a more reliable method to validate and biochemically dissect the transport mechanism of a transporter is to purify and reconstitute its activity in vitro.

Reconstitution of transmembrane proteins can be divided into two broad classes—symmetric, where both leaflets of the bilayer are accessible such as planar bilayer reconstitution of ion channels, and asymmetric, such as liposomal reconstitution, which mimics a cellular compartment and has a distinct luminal side (21). Liposomes have been widely utilized as the model system for studying membrane transporters in a purified system because they mimic natural cell membranes in several different ways. Liposomes form in aqueous solution when the polar head groups of phospholipids encounter water and due to persistent entropic challenge, the molecules are forced to arrange themselves in a system of closed membrane where the polar heads are exposed to the aqueous phase while the hydrophobic tails are buried within the core. Liposomes were first described by Bangham in the mid 1960s (22) and have become one of the greatest tools for controlled and targeted drug delivery applications. The first application of liposomes to study the biological functions of membrane proteins was the reconstitution of ATP-Pi exchange catalyzed by the mitochondrial ATP synthase (23), followed by cytochrome oxidase proton transport (24) and ATP-dependent calcium transport by Ca2+/Mg2+-ATPase (25). From then on, the use of proteoliposomal reconstitution has grown over the years for characterization of the functions and mechanisms of transmembrane proteins.

Liposomes are produced by dissolving desired lipids in an appropriate buffer to generate multiple layers of concentric membranes that are subsequently broken into smaller unilamellar structures upon sonication. The liposomes are then destabilized by adding detergents at concentrations much below the critical micelle concentration. Mixing the destabilized liposomes with detergent solubilized protein allows the protein to partition in the lipid bilayer. The final step involves detergent removal using absorptive agent, gel filtration, or dialysis (Fig. 1) (26, 27). The schematic in Figure 1 illustrates the reconstituted protein in one orientation, but in reality proteoliposome reconstitution of transmembrane proteins can result in a mixture of inside-out and outside-out orientations (28, 29). We will discuss this in more details later.Figure 1 Schematic overview of protein extraction, purification and proteoliposome reconstitution. Detergent is used to solubilize and extract membrane proteins, and the desired protein is separated from the bulk of endogenous host proteins by a combination of different chromatographic methods to achieve desired purity. Liposomes are destabilized with detergent to yield detergent-containing liposomes. The detergent-solubilized protein is mixed with destabilized liposomes followed by detergent removal to yield proteoliposomes.

For studying metal transport assay, typically a metal-sensitive fluorescent probe like Phen Green SK, calcein, or Fura-2 is mixed with the reconstituted proteoliposome and subjected to freeze-thaw cycles to internalize the probe. Freezing and thawing creates unrestricted movement of water in between the polar headgroup, allowing the water-soluble dye to be entrapped in the aqueous lumen when the proteoliposomes reform (Fig. 2A). The solution is extruded through 0.4 μm filters to generate unilamellar vesicles which are then purified via a gel filtration column to obtain pure dye-encapsulated proteoliposomes. Adding a suitable metal ion substrate to the proteoliposomes triggers the transport of the metal ion into the lumen mediated by the transporter incorporated in the lipid bilayer. As the transported metal interacts with the fluorescent probe, the change in the fluorescence intensity of the probe (Fig. 2B) can be used to measure metal ion transport. The influence of voltage on the transport kinetics can also be assessed by diluting dye-loaded proteoliposomes into buffer containing appropriate concentration of KCl to establish the desired potential across the proteoliposome bilayer (Fig. 2C). This is a well-established method where a K+-selective ionophore like valinomycin utilizes unequal concentrations of K+ ions on the inside and outside of the proteoliposome membrane to generate a voltage difference across the membrane (30). For metal transporters that exhibit voltage-dependent uptake/efflux, the transport rate will increase with increasing negative potential as illustrated in Figure 2D. As we will discuss later, this technique has been used to demonstrate interesting features of metal transport catalyzed by DraNramp (31).Figure 2 Metal ion transport by Proteoliposomes.A, schematic overview of proteoliposome-dependent fluorescence quenching assay. B, illustration of time-dependent fluorescence measurement during a transport assay. The fluorescence of a metal-sensitive dye internalized within the proteoliposomal lumen is quenched upon interactions with metal ions that move from the outside into the lumen. C, schematic representation of proteoliposome metal uptake assay conducted at variable membrane potentials (ΔΨ). Membrane potentials are usually established using K+ gradients and valinomycin. Fura-2 is a metal-sensitive dye used to detect metal transport. D, representative time traces of metal ion transport at different ΔΨs. A more negative membrane potential accelerates metal transport rates by DraNramp (31).

The advantage of the reconstitution approach is that every component of the system including the choice of lipids, ionic composition, and the protein are under the control of the experimenter. This is distinct from the complex environment of the cell comprising various small molecules, membranous structures, cytoskeleton, and of course, thousands of proteins. However, it is useful to reiterate that the size of proteoliposomes used in reconstitution experiments differ by several orders of magnitude compared to a living cell. The nature of interactions, complex formation and molecular dynamics can also be very dissimilar since the morphological properties and composition of the lipid bilayer are very distinct. Therefore, comparing findings from in vitro reconstitution studies with in vivo experiments, and to verify the biological significance of the findings, is an essential step to mitigate potential erroneous conclusions. As an example, MavN (more regions allowing vacuolar colocalization N (32)) is a protein encoded by the intracellular bacterial pathogen Legionella pneumophila which is inserted in the membrane of the compartment in the host cell where the bacteria proliferate (33). It serves as a conduit for the trafficking of transition metal ions from the cytoplasm of host cell into this compartment. A Cys301Ala mutation in MavN exhibited partial defect in iron transport in vitro, which would have been predicted to be a loss of function mutation, but the same mutant promoted intracellular proliferation of Legionella (34). This suggests that the mutant retains its affinity to substrate but possesses inefficient substrate release capabilities which may be potentiated by other factors in the native environment.

Overcoming the challenges of purification and proteoliposome reconstitution

The instability of eukaryotic transmembrane proteins is one of the major technical hurdles that must be addressed prior to interrogating the function of any transition metal transporters in vitro. This challenge is not specific to transition metal transporters per se but is common to all eukaryotic transmembrane proteins (35). Once they are extracted from the membrane, they are generally very fragile and need to be handled carefully. Transition metal transporters consist of at least six transmembrane helices embedded in the lipid bilayer. Typically mild nonionic detergents are used to solubilize the protein of interest from the lipid bilayer prior to purification. There is no “one size fits all” rule when it comes to selecting a detergent because different proteins behave differently in specific detergents. Therefore, selecting a detergent that prevents aggregation and enhances protein stability is a crucial step in the purification of membrane proteins. Mild, nonionic detergents have been used to successfully determine the structures of a few transition metal transporters (Table 2). Prior to that, ortholog screening may be a necessary first step to identify a construct with improved expression level, on the conceptual basis that the inherent variation in the primary sequence between orthologs of the same protein result in a few members with better biochemical stability and thus a high production yield in a recombinant system, to facilitate biochemical and biophysical studies (36). The orthologs are typically fused to GFP or its variants (37) for easy analysis via fluorescence-detection size-exclusion chromatography (38). The advantage of ortholog screening is best illustrated with the example of natural resistance–associated macrophage protein (Nramp), a divalent transition metal transporter. Although the first high-resolution structures of Nramp were solved by the Dutzler lab for the Staphylococcus capitis (ScaDMT) ortholog, the authors subsequently used the Eremococcus coleocola (EcoDMT) ortholog for functional characterization since the latter had faster kinetics (10).Table 2 Choice of detergent for structural studies

Protein	Initial detergent	Final detergent	Structural method	Reference	
DraNramp	DDM	DM or LMNG	X-ray crystallography	(28, 37)	
FPN	DDM, CHS	DDM, CHS	Cryo-EM	(38)	
ScaNramp	DM	DM	X-ray crystallography	(39)	
YiiP	DDM	DDM	X-ray crystallography	(40)	
EcoNramp	DM	DM	X-ray crystallography	(10)	
TsFPN	LMNG	DDM	X-ray crystallography	(41)	
BbFPN	DDM	LMNG	X-ray crystallography	(42)	
BbZIP	DDM	DDM	X-ray crystallography	(43)	

Addition of lipids to purification buffers may also enhance activity and yield of desired product. For example; including tetraoleoyl cardiolipin, which is widely regarded as a major component of mitochondrial membranes, in purification buffers improved the final yield and activity of the mitochondrial iron importer mitoferrin 1 (39). Adding zinc chloride to the purification buffers at pH 6.0 yielded a more stable sample of a bacterial homolog of ferroportin from Bdellovibrio bacteriovorus (BbFPN) that generated larger crystals in comparison to BbFPN at pH 8.0, lacking zinc chloride (40). While the presence of Zn2+ ions in the buffer could interfere with functional analyses, it was interesting to observe that the latter buffer system yielded another crystal form that was not apparent in protein samples purified in the presence of zinc chloride (40). In the absence of substrate, Zrt- and Irt-like protein (ZIP) from Bordetella bronchiseptica (BbZIP) was largely prone to aggregation but the phenomenon was significantly mitigated when cadmium ions were present in the purification buffers, leading to high-yield homogenously pure sample (41). As illustrated in these cases with transition metal transporters, these studies demonstrate that substrate addition may offer some benefits with respect to protein stability. It is useful to point out here that use of metal affinity chromatography can complicate metalation states of the transporters, an aspect that likely varies on a case-by-case basis.

Reconstitution of transmembrane proteins into proteoliposomes, although a well-established technique, can be fraught with challenges. Intrinsic leakiness can occur in poorly formed proteoliposomes where the intended substrate may passively move across the lipid bilayer without the need of a transporter. This situation is mostly influenced by one, or a combination of four factors— oxidation of lipids by metal ions, high protein to lipid ratio, lipid composition of the liposome, and the use of high substrate concentrations. For reconstitution of transition metal transporters with redox active substrates, oxidation of lipids presents one of the most critical challenges in maintaining the integrity of proteoliposomes. Strategies to overcome this limitation usually involve supplementing the assay mixture with additives (please see below) that prevent lipid oxidation but do not form strong complexes with the substrate under assay conditions, that would preclude uptake of the substrate by the transporter under investigation. Selecting the right choice of lipids through empirical evaluation, determining the optimal protein to lipid ratio, as well as using low substrate ion concentrations are also crucial to obtain good results. In the case of EcoNramp, the authors discovered that use of synthetic phospholipids, particularly a 3:1 mixture of POPE:POPG led to a tighter proteoliposomal reconstitution compared to reconstitution in native lipid mixtures. It was also essential to use a low protein:lipid ratio (10, 42).

EDTA is usually used to remove residual labile metal ions prior to performing transport assay, but it is important to keep in mind that prolonged EDTA incubation may also cause irreversible aggregation as was the case with YiiP (43). This can happen if a tightly bound metal ion substrate is carried through the purification by the transporter and the displacement of that metal ion causes destabilization of the protein. Treatment of assay buffers with Chelex resin to remove trace divalent ions has been shown to reduce background noise in the proteoliposome-reconstituted metal ion transport assay (39). Other components of the assay buffer can also play an important role toward a successful in vitro reconstitution of metal ion transport. For example, Mops buffer is known to stabilize Fe2+ for a considerable length of time in solution (44). Including additives such as sorbitol which scavenges reactive oxygen species, or histidine which protects liposomes against copper-induced damage, to the assay mixture would significantly influence a successful transport activity measurement with iron or copper as respective substrates (39).

In vitro reconstitution of the activities of transition metal ion transporters

Substrate-bound structures of metal transporters are scarce, making it challenging to discern comprehensive mechanistic insights from atomic resolution structures alone. Thus, the importance of biophysical and biochemical analyses for gaining mechanistic insights cannot be overstated. A couple of studies aiming to understand the mechanisms of transport and substrate recognition/selectivity are consistent with the classical alternating access transport mechanism (see Table 1). In this mode of transport, the binding of metal ion to the binding site induces structural rearrangements within the transmembrane helices causing the transporter to cycle through at least four different conformational states, as the substrate navigates through the transport pathway (Fig. 3A). Interestingly, transition metal transporters exhibit very distinct transport mechanisms. The three mechanisms reported thus far are uniport, symport, and antiport, which may or may not be proton dependent (Fig. 3B). A uniporter transports one ion in one direction whereas a cotransporter utilizes the free energy generated by the downhill flux of one ion to drive the movement of another ion in the same direction (symporter) or in opposite direction (antiporter).Figure 3 Schematic representation of transport mechanism and putative conformational states of metal transporters. (A) classical alternating access transport mechanism and (B) four different transport mechanisms exhibited by transition metal transporters. Two additional binding sites have been shown to include sites where the metal binds to the protein on either side of the membrane before and after the transport.

Zinc transporters

Chao and Fu reported the first in vitro reconstitution of the Escherichia coli cation diffusion facilitators, ZitB and YiiP (43, 45). Purified YiiP was studied using isothermal titration calorimetry (ITC), and the findings revealed a mutually competitive binding site that is common for Zn2+, Cd2+, and Hg2+. By using stopped-flow measurements of metal ion flux across proteoliposomes encapsulating a metal-sensitive fluorescent dye, they showed that both ZitB and YiiP are proton-dependent antiporters facilitating the exchange of Cd2+ or Zn2+ for H+, in a 1:1 stoichiometric fashion. ZitB-mediated transport is a substrate-saturable process involving a two-step reaction—the initial binding of the metal ion followed by a conformational rearrangement that helps to move the substrate across the membrane. Proton influx downhill generates the free energy needed to drive cytoplasmic zinc efflux (45). This study also identified a set of noncompetitive binding sites for each of the three metal ions and showed that Zn2+ binding partially inhibited the bindings of both Cd2+ and Hg2+, as well as the role of histidine residues in binding-deprotonation coupling mechanism (43).

The crystal structure of YiiP revealed a homodimer with four coordinating Zn2+ ions, holding the two monomeric subunits in a parallel orientation at a densely charged cytoplasmic interface, while the two six-transmembrane helical domains adopt a Y-shaped structure (Fig. 4A) (46). A subsequent X-ray crystal structure suggested that the transmembrane domains of YiiP are spread out in a manner that prevents intermolecular contacts (47), whereas a cryo-EM structure showed close contact between the transmembrane helices (48). To investigate the functional consequences of this discrepancy, the Stokes lab used cysteine crosslinking to constrain the domains in a compact state (48). They then performed in vitro proteoliposome reconstituted Zn2+ transport assays with these mutants, revealing that the splaying apart of the domains did not influence transport activity (48).Figure 4 Atomic resolution structures of transition metal transporters. In complex with A–D, substrates, B, a modulator and E, an inhibitor. Gold color indicates TMs that contributes to the molecular architecture of the transport pathway.

The cytoplasmic domain of each protomeric unit in the dimeric complex has metallochaperone-like protein fold, while the tetrahedral Zn2+ binding sites form a cavity that extends to both intracellular and extracellular side of the membrane (46). The structure also revealed the residues that coordinate Zn2+, to be consistent with the binding site residues identified in the proteoliposome-reconstituted transport assay involving YiiP mutants (43, 45). Lu et al., identified a conserved salt bridge at the cytoplasmic membrane surface which they suggested to contribute critically to dimer formation, and mediate zinc-induced interdomain movements during transport (49). However, using UV, multiple-angle light scattering and refractive index detectors, the size-exclusion chromatographic profiles of two mutants that disrupted Zn2+ binding at the cytoplasmic domain interface revealed that those binding sites did not affect dimer formation (48). This demonstrated that the YiiP dimer is extremely stable, suggesting potential existence of other unknown features at the cytoplasmic interface that mediate dimerization. More studies would be needed to uncover them.

Further investigation using site-specific mutagenesis, stopped-flow spectrofluorometry and isothermal titration calorimetry identified a highly conserved Aspartate residue (Asp157) in YiiP that is responsible for its selectivity for Zn2+ and Cd2+ (50). Functional and structural analyses of purified YiiP using maleimide polyethylene-oxide biotin labeling and chemical cross-linking identified yet another highly conserved aspartate residue (Asp49) that contributes to a common Cd2+ binding site, underscoring a unique coordination chemistry between metal ions in the translocation channel of YiiP and aspartate residues (51). Structure-function analysis of an improved structure of YiiP (2.9 Å resolution) confirmed that Asp157 and Asp49 are involved in coordinating Zn2+ at a conserved binding site located within the transmembrane domain (49). Putting together the data, a zinc-regulated zinc transport mechanism was proposed in which the coordination chemistry of the four binding sites is allosterically modulated by movements between the two C-terminal domains, triggered by rising concentrations of zinc in the cytoplasm. As a result, the efflux activity of YiiP is reinforced to counter the accumulation of excess zinc. These were the first structural details of how metal transport activity is regulated by metallochaperone-like cytoplasmic domain. The metallochaperone-like cytoplasmic domain is a common theme (52) and its regulation of metal coordination chemistry in response to cytoplasmic metal ion imbalance may offer a plausible regulatory mechanism for metal transporters. Nevertheless, in a recent study, multiple independent cryo-EM maps were compared, suggesting a mechanism where Zn2+ binding at the secondary site at the cytoplasmic membrane interface mediates a transition from the inward-facing state to an intermediary conformation between the inward-facing and outward-facing states (53).

The zinc transporter-8 (ZnT8) is an important element of the insulin secretory pathway and primarily sequesters Zn2+ in the insulin-secretory granules of pancreatic β-cells (54, 55). The Fu lab reconstituted ZnT8 into proteopliposomes and showed that clinically relevant variants of the transporter are functionally modulated by lipids (56). They investigated the influence of the three major classes of lipids in insulin granules namely anionic phospholipids, nonbilayer phospholipids and cholesterol, on the transport activity of human ZnT8 variants. The rationale for this investigation stemmed from the fact that cholesterol alone accounts for 20 to 25% composition of the insulin secretory granule membrane (57), while lysophosphatidylcholine makes up to 20% of the total granule lipid content (58). The Fu lab demonstrated that adding anionic phospholipid enhanced zinc transport capacity of ZnT8 irrespective of the chemical nature of the lipid headgroup, whereas adding lysophosphatidylcholine or 5% cholesterol to the proteoliposomes strongly inhibited the activity of the transporter (56). Since both cholesterol and lysophosphatidylcholine are nonbilayer lipids, they may partition between the granular membrane and residential lipid-binding proteins to influence the activity of ZnT8 in the insulin granule membrane. The type 2 diabetes high-risk variant (Arg-325) of ZnT8 exhibited higher transport activity in comparison to its low-risk counterpart (Trp-325) (56), underscoring a therapeutic strategy where the hyperactive variant may be targeted for inhibition to reduce type 2 diabetes risk.

Iron transporters

In eukaryotes, mitochondria play a centrally important role in the cell biology of iron (59) and the only known major mitochondrial iron importers are mitoferrin-1 (Mfrn1) and mitoferrin-2 (Mfrn2). For more than 2 decades following the discovery of DMT1, iron was largely (and is still) regarded a troublesome substrate due to its intrinsic properties that pose major challenges to in vitro transport assays using proteoliposome reconstituted metal transporters. The ability of the metal to catalyze lipid peroxidation and the fact that it subsequently precipitates upon oxidation from Fe2+ to Fe3+ leads to leaky liposomes (60). Some of the same underlying mechanisms were also discovered in the context of the ferroptosis pathway (61).The first ever bona fide proteoliposome reconstituted iron transport assay was reported by our lab, demonstrating the iron transport activity by Mfrn1 from Oreochromis niloticus (TMfrn1). There were several key components to the successful establishment of this assay—one of them was the addition of a chelator to the assay buffer that rigorously removed other transition metal ions and prevented spurious quenching of the fluorescence reporter. Another was the addition of sorbitol, which presumably stabilized the iron in its Fe2+ form and prevented lipid oxidation and thus loss of liposome integrity. Mitoferrin had been proposed to be an iron transporter (62) but this study conclusively demonstrated its iron transport activity with purified protein. Mitoferrin is a mitochondrial inner membrane protein and addition of cardiolipin, which is a critical component of mitochondrial membrane, was essential during both purification as well as successful reconstitution of metal transport activity of Mitoferrin.

A combination of ITC experiments, proteoliposome-reconstituted assay, and site-directed mutagenesis was used to describe a detailed biochemical and biophysical behavior of the transporter. ITC data showed that Mfrn1 has high binding affinities to Fe2+, Mn2+, Co2+, and Ni2+, while data derived from proteoliposome-reconstituted transport assay showed that Mfrn1 transports Fe2+, Mn2+, Co2+, Cu2+, and Zn2+, but was robustly inhibited by Ni2+ (39). This assay was used to carry out mutational analyses of transport function, which identified important cysteine, histidine, and methionine residues that are critical for substrate binding and transport activity of Mfrn1. Although several mitochondrial solute carriers exhibit a substrate-proton symport mechanism, in vitro reconstitution data for Mfrn1 did not indicate a proton-coupled transport mode (39), suggesting a potentially different transport mechanism. Nevertheless, additional analysis is required to determine whether Mfrn1 cotransports or exchanges Fe2+ with another substrate.

This assay was subsequently used by our lab to show that MavN (32), an effector protein encoded by the intracellular bacterial pathogen L. pneumophila (63) is a bona fide iron transporter. In combination with cell-based experiments, ITC measurements and mutagenesis, we showed that MavN can also transport Mn2+, Co2+, and Zn2+, but not Cu2+ and Ni2+ (34). Mutational analyses identified residues such as Glu439, His412, His445, Met204, and Cys301 that are important for binding and transport of metal ions. In a remarkably striking correlation between in vitro and in cellulo data, a MavN-deficient strain of Legionella harboring plasmid encoding the respective mutants restricted or nearly abolished growth (34). These findings highlighted the mechanism by which Legionella, and perhaps other opportunistic bacteria, acquire the essential trace metal nutrients for growth and proliferation.

Ferroportin (FPN) is the only known iron exporter found in vertebrates that catalyzes the release of iron from cells that regulate iron utilization and storage, into blood plasma (64). Hepcidin is a peptide hormone that regulates FPN activity by binding and mediating internalization and subsequent degradation of the exporter (65, 66, 67). Hepcidin regulation of FPN is the major systemic iron homeostatic control strategy (68). In order to dissect the molecular basis of iron transport by FPN, purified human FPN1 was incorporated into proteoliposomes. Transport assays showed that the protein prefers Fe2+ as a substrate while also exhibiting reduced affinities for other divalent metal ions such as Co2+, Ni2+, and Mn2+ (69). The FeCl2 solution used in the transport assay was supplemented with excess sodium ascorbate to prevent oxidation of Fe2+ to Fe3+ (69, 70). Human FPN1 is a proton-coupled symporter that can catalyze the active efflux of Fe2+ against Fe2+ concentration gradient, driven by favorable pH gradients. Interestingly, in a different study, the activity of FPN from Tarcius syrichta (TsFPN) was reconstituted in a proteoliposome based assay with purified protein that did not necessitate adding any stabilizer for Fe2+. TsFPN demonstrates a different mode of transport where the transport of Fe2+ is coupled to the transport of two protons in the opposite direction (antiporter) (70). A bacterial homolog, BbFPN was reconstituted into liposomes and Fe2+ influx measurements were performed in the presence of sodium dithionite, a reducing agent, diluted in the assay buffer immediately prior to the assay to avoid the oxidation of Fe2+ (40). The data were consistent with a uniporter-like transport mode, in which BbFPN moves Fe2+ and other divalent metal cations along their concentration gradients (40). Based on the disparities in the mode of transport for these three transporters, one may be tempted to assume a species-dependent activity mechanism; however, additional experiments will be needed to shed light on these differences.

Structural characterization of BbFPN revealed intrinsic conformational rearrangements during its transport cycle that involves transmembrane helix bending (40). The study also predicted a putative hepcidin binding site in the central cavity of the transporter which has subsequently been validated by a cryo-EM structure of lipid nanodisc-reconstituted human FPN in complex with hepcidin and Co2+ (71). The cryo-EM structures of both the hepcidin-bound (Fig. 4B) and the apo-FPN revealed binuclear binding sites located within the N-terminal and C-terminal domains. The binding of hepcidin to an outward-open FPN is coupled to Fe2+ binding to the C-terminal domain, resulting in a complete blockage of the efflux pathway and inhibiting the activity of metal transport by FPN. Since iron binding increases the affinity of hepcidin to FPN by 80 fold (71), it is reasonable to suggest that only iron-bound FPN would preferentially bind hepcidin and therefore be targeted for biotinylation and subsequent degradation.

Manganese transporters

DMT1 belongs to the family of Nramp2 which is largely expressed in the duodenum, placenta, brain, kidney, and testis (8) where it contributes primarily in nonheme iron uptake (72). DMT1 homolog, Nramp1, is natively expressed in macrophages, where it typically functions in host defense against pathogens (73), with manganese as its physiological substrate (1). The first crystal structure of the Nramp1 family member from S. capitis (ScaNramp) was determined almost a decade ago (74), followed by DraNramp from Deinococcus radiodurans (28, 75) and EcoNramp from E. coleocola (10, 76). Divalent metal ion transport by ScaNramp was characterized using a fluorescence-based assay by reconstituting into proteoliposomes composed of a 3:1 mixture of E. coli polar lipid and L-α phosphatidylcholine. The authors specifically noted the incompatibility of the transport assay with Fe2+, Cu2+, or Pb2+. The slower kinetics of ScaNramp and the less stable proteoliposomal reconstitution with the native lipids necessitated honing the reconstitution with synthetic lipids and EcoNramp, which has a faster kinetics, for subsequent characterization of Mn2+ transport by this transporter (10). The Dutzler lab subsequently used this assay to demonstrate that transport of metal was coupled with H+ transport by using the pH sensitive fluorophore 9-amino-6-chloro-2-methoxyacridine. This proteoliposomal reconstitution system enabled the authors to identify, together with crystal structures, the metal binding residues as well as residues involved in proton transport.

For DraNramp (74), inconsistencies between experimental data from the transporter expressed in E. coli and that from proteoliposome reconstituted system in vitro led to the investigation into the role of transmembrane voltage on the transporter’s function using an in vitro proteoliposome reconstituted assay. The Gaudet lab noticed that purified DraNramp reconstituted into proteoliposomes only transported Cd2+, but not physiologically relevant substrates such as Mn2+ or Fe2+, or biologically relevant metals such as Zn2+ or Co2+ (31). An equimolar solution with ascorbic acid was used for stabilizing the Fe2+. They then used a well-established method of using a K+-selective ionophore and starting with unequal concentrations of K+ on either side of the liposome to create a voltage difference across the membrane in their proteoliposomal system (30). This enabled them to interrogate the transport as a function of voltage difference across the membrane. Intriguingly, DraNramp was able to transport the biological metal substrate at rates between −40 mV and −80 mV with noticeable potential difference requirements in the order Mn2+ < Zn2+ < Fe2+ < Co2+, and the Michaelis–Menten constant (Km) values were consistent with those obtained from in vivo metal uptake experiments (31). These findings indicated that transmembrane voltage is a crucial variable that regulates the kinetics of metal transport by the Nramp family of transporters.

Interestingly, the Nramp1 family seems to deviate from the basic transport mechanism exhibited by a canonical symporter that catalyzes proton-metal cotransport in that the substrate type influences the stoichiometry of transport, allowing the transporter to function as a voltage-dependent proton-metal symporter, metal uniporter, as well as proton uniporter (31). Proton uniport and metal transport occurs via distinct conformational states of the transporter. In an elegant application of the proteoliposomal reconstitution assay, this was demonstrated by investigating transport property of site directed cysteine mutants. Post reconstitution, modification of the cysteines by positively charged 2-(trimethylammonium)ethyl methanethiosulfonate abolished metal transport but retained proton transport. However, modification by neutral 2-aminoethyl methanethiosulfonate or N-ethylmaleimide moderately impaired metal transport activity. There is a network of conserved salt-bridges that establish a proton-transport pathway extending from the metal-binding site to the cytosol, which if perturbed, leads to a multitude of unwarranted transport behavior of the DraNramp under physiological-like conditions, underpinning the importance of this structural feature in supporting efficient metal transport (31). The conserved molecular mechanism, conformational rearrangements, metal ion binding and release, substrate identity, proton transport, voltage dependence, and proton-metal coupling of the Nramp family of membrane transporters have been extensively reviewed by Bozzi and Gaudet (1).

Metal ion influx by Nramp appears to be not thermodynamically dependent on the concentration gradients of other metal ions or protons, to electrochemically power the uphill movement of target substrate. Influx occurs much more readily than efflux, suggesting that Nramp structure may have evolved to establish a strong kinetic barrier that preserves cytosolic metal stores. One aspect that remains unclear is whether proton uniport, which appears to be a common phenomenon in the Nramp family, is an inefficient use of electrochemical energy since the transmembrane proton gradient is dissipated without contributing to metal uptake. Nevertheless, it is also likely that the proton uniport may constitute functional advantages that are yet to be determined. Additional studies are needed to establish whether the transport events carried out by the Nramp family follow a thermodynamically coupled metal-proton symport system, and to uncover the mysteries governing metal-binding induced conformational rearrangements, that may perhaps influence proton movement via the salt-bridge network.

A total of 14 Nramp structures at different conformational states including inward-open, outward-open, and occluded conformations, have been resolved (1); three of them have Mn2+ bound to the metal binding site, including ScaNramp (Fig. 4C) and DraNramp (Fig. 4D) (10, 28, 74). Another study reported the crystal structure of EcoNramp in complex with a brominated analog of bis-isothiourea (Br-BIT), an inhibitor bound to its extracellular binding pocket (Fig. 4E), preventing substrate loading (76). The structures of transporter–substrate complexes have significantly helped in understanding aspects of substrate selectivity and specificity. Substrate selectivity is another intriguing topic in transition metal transport that is not well understood, but a recent study attempted to fill this knowledge gap by resolving the crystal structures of metal bound DraNramp at three different stages of the transport cycle. The study revealed distinct coordination geometries for Mn2+ and Cd2+ that induce global conformational changes as the substrates move through the transport pathway from the outer to the inner gate (77). Multiple studies have reported that both outer and inner gates are lined by conserved networks of polar residues that establish a gate/latch interface to achieve alternating access. The availability of structural data together with biochemical evidence have enabled a full illustration of the transport cycle for Nramp metal ion transporters, detailing the distinct roles of substrate binding, release, and the conformational changes occurring within the transmembrane helices to facilitate the transport of both proton and metal ions.

Copper transporters

The copper transporting P-type ATPases regulate copper homeostasis in all kingdoms of life by pumping Cu+ ions across cellular membranes (78, 79, 80, 81). A combination of high-resolution structures and molecular dynamics simulations delineated the transport pathway of Cu+-transporting P1B-ATPases and provided important information that improved our understanding of the pathogenesis of certain Menkes' and Wilson's disease mutations (82, 83, 84, 85, 86). Although these studies established the structural details of the transport function of Cu+ pumps, functional reconstitution of Cu+ transport using purified protein was still lacking until recently when the Meloni lab reconstituted the Cu+-pump from E. coli (EcCopA) into small unilamellar proteoliposomes and quantitatively characterized its metal ion substrate translocation (87). Prior to this work, the Xiao lab had reconstituted EcCopA into giant unilamellar vesicles and used 1H nuclear magnetic resonance spectroscopic approach to demonstrate a direct correlation between Cu + transport and the ATPase activity of EcCopA (88). However, three important questions remained unanswered—(i) whether Cu+-pumps are uniporters or cotransporters; (ii) information about their electrogenicity; and (iii) establishing a full understanding of the aspect of ATP-dependent Cu+ transport. The Meloni lab addressed these questions by establishing an assay that allowed real-time measurement of substrate transport, counter-ion movement, and charge translocation of EcCopA incorporated into proteoliposomes (87). To achieve this, they encapsulated the lipid-compatible Cu+-selective fluorescent probe CTAP-3 (89) in the proteoliposome lumen and determined the transport kinetics of EcCopA, revealing that Cu+-ATPases are electrogenic uniporters as opposed to other P-type ATPases like Ca2+/H+ pump that exchange H+ ions. By encapsulating the pH sensor pyranine (90) and the membrane potential sensor oxonol VI (91) in the lipid vesicles, the authors also showed that EcCopA transports one positive charge per ATP hydrolysis cycle to build up transmembrane potential. To maintain liposome integrity and prevent copper-induced lipid oxidation, stock solutions were freshly prepared in an anaerobic glove box purged with constant flow of nitrogen prior to each experiment, and the proteoliposomes and control liposome solutions were supplemented with ascorbic acid (87).

Orientation of reconstituted transporters in liposomes

One unavoidable aspect of bottom-up reconstitution of purified membrane proteins in detergents into proteoliposomes is that the transporter can get inserted in both orientations. Intriguingly, this is not always 50:50 and there seems to be a protein dependence on the ratio between the two possible orientations in the proteoliposome population. This brings up three follow up queries: (1) how to experimentally assess the distribution of the two orientations; (2) how to separate them from each other, if possible and (3) if it is feasible to interrogate the function of transporters oriented in only one orientation.

In the context of transition metal transporters, the ratio of the two orientations was experimentally determined by the application of an earlier strategy developed by the Miller lab (42, 92). This method relies on the protection afforded by the internal side of the liposome from reagents added to the external side. Thus, a protease cleavage site can be accessed and thus cleaved by added protease, in one orientation of the transporter but not the other. In the presence of excess detergent, however the liposome breaks down, and both sides can be accessed. This method was used to experimentally determine the ratio of the two orientations of proteoliposome reconstituted DraNramp by the Gaudet lab (28) and that of EcoDMT1 by the Dutzler lab (76). The same principle was used by the Miller lab to also develop a method for interrogating selectively the function of the transporter in one orientation. Here, a functionally innocuous cysteine is engineered in the transporter which in one orientation faces the external side of the liposome and thus can be modified by membrane impermeant cysteine modifying reagents that can add positive charge (2-(trimethylammonium)ethyl methanethiosulfonate) or negative charge (MTSES) and can render the modified transporter inactive. In the other orientation, the cysteine is shielded by the liposome membrane and is thus not accessible and is not modified. Thus, post modification, the transporters facing one orientation are “functionally silenced”. This strategy was used to interrogate the residues separately involved in metal transport versus proton transport for DraNramp (28).

For membrane transporters in general, proteo-lipobeads are being investigated as a plausible solution to the orientation issue and perhaps represent a very promising tool for future development (93). In this technique, the His-tagged membrane protein is bound to Ni-NTA-functionalized beads prior to reconstitution, allowing a continuous liposomal membrane to form around the bead-support (94, 95, 96). The Jiang lab mixed purified detergents-solubilized voltage-dependent K+ channel (KvAP) with Ni-NTA functionalized silica beads in the presence of lipids, and then slowly removed the detergents using Bio-Beads, reconstituting a bilayer membranes around the surface-anchored channels with near 100% unidirectional orientation (94). Similar results were also obtained for the reconstitution of cytochrome c oxidase (95, 96). This technique has not yet been attempted with preformed liposome which could offer interesting applications.

Another proof-of-concept example was reported, where the researchers manipulated the overall charge distribution across the lipid bilayer surface to influence the directionality of proteoliposome-reconstituted proteorhodopsin. By mixing lipids with different charge properties, the Ajo-Franklin lab showed that proteorhodopsin orientation was largely determined by the type (cationic or anionic) of liposomes used (97). Interestingly, the Müller lab introduced another method to direct the orientation of proteorhodopsin where they explored the fact that soluble proteins would not readily cross the lipid bilayer to generate N- and C-terminal fusion constructs of the proton pump (98). Using this strategy they demonstrated that the position of the soluble fusion partner exclusively determined how the membrane protein inserted into preformed vesicles (98). The MacKinnon lab recently reported another approach where they isolated small vesicles containing the calcium-activated potassium channel, Slo1, from its native membrane environment without the use of detergents (99). The Slo1 construct was engineered to contain an intracellular-oriented C terminally fused GFP. Vesicles containing Slo1 with an inside-out orientation were then separated from the bulk of small unilamellar vesicles using GFP nanobody affinity purification (99). This technique can be particularly useful when studying the biochemical characteristics of membrane transporters that may have cofactors, weakly interacting proteins or lipids that participate in their biological function.

Targeting transition metal transporters for future therapeutic development

Dysregulation of trace metal homeostasis is associated with the pathogenesis and progression of several diseases including cancer and neurodegenerative diseases. The prevalence of treatment resistance especially in cancer has incentivized the development of new strategies to eliminate these bottlenecks and improve treatment outcomes. Among the few approaches are transition metal chelators and ionophores that are designed to selectively alter the concentrations of iron, zinc, and copper in cancer cells. Although several of these agents have demonstrated appreciable targeted activity against drug-resistant and cancer stem cells, devising a formulation technique that would enable precise targeting has remained a major issue (100, 101).

Targeting the transport of transition metals is a plausible approach to address diseases that are caused by their misregulation. Examples of transporters that have been suggested for specific pathologies include DMT1 in Parkinson disease (102); ZIP8 in osteoarthritis pathogenesis (103); mitoferrin 1 in Friedreich’s ataxia (104, 105); mitoferrin 2 in Huntington’s disease (106); and FPN in β-thalassemia (107).

Chemical discovery efforts have generated a repertoire of small molecule inhibitors against DMT1 (108, 109, 110). Some DMT1 inhibitors target cancer stem cells by blocking lysosomal iron translocation, leading to iron accumulation in the lysosomes, triggering formation of reactive oxygen species and ultimately cell death (111). Other studies indicate that DMT1 regulates interactions between the mitochondria and early endosomes to facilitate iron translocation into the mitochondria (112), and mitochondrial iron overload promotes development of malignant features in cancer cells (113). Therefore, inhibiting DMT1 could be a plausible strategy for developing novel cancer therapeutics.

In vitro reconstitution systems offer a useful alternative to screen for potential new drug candidates as well as evaluation/validation of existing ones. For instance, the in vitro reconstitution of the transport activity of EcoNramp provided useful insights on how analogs of bis-isothiourea may be exploited as potential therapeutic strategies against human DMT1 in pathologies associated with iron overload such as hemochromatosis, and for laying down a framework for optimization to achieve higher activity and selectivity (76). In vitro binding and transport experiments demonstrated that hepcidin binds and occludes FPN from the extracellular space to inhibit its transport activity, and also indicated that the binding of hepcidin is mediated by the presence of divalent metal (71). These functional insights can be exploited to benefit human iron disorders such as anemia. A good example of such effort is the small molecule inhibitor vamifeport which outcompetes hepcidin binding of FPN (114), and is currently undergoing clinical development for treatment of β-thalassemia and sickle cell disease (115, 116). Studies conducted in mouse models demonstrated that minihepcidins are promising therapeutic candidates against human diseases like β-thalassemia and hemochromatosis (117, 118). In vitro proteoliposome-reconstituted transport activity assay was recently used in conjunction with structural information to reveal novel interactions between FPN and a nanopeptide mimetic of hepcidin named PR73 (119), providing new insights that would potentially guide future therapeutic development aiming at increasing inhibition potencies of FPN inhibitors.

Transition metal transporters also have the potential to serve as biomarkers for early detection and prevention of diseases. For example, the Fu lab recently identified a new islet autoantibody named ZnT8ecA directed to the extracellular epitopes of the zinc transporter ZnT8 (ZnT8ec) in patients with type 1 diabetes (120) and in a mouse model of type 1 diabetes (121). They showed that ZnT8ecA interactions with ZnT8ec cause immunological stress in β-cells which are believed to be a hallmark of type 1 diabetes pathogenesis (122). They further generated and characterized a high-affinity monoclonal antibody (mAb43) that specifically targets both human and rodent ZnT8 and used it to uncover a functional correlation between glucose-stimulated insulin secretion and mAb43 binding to ZnT8 on β-cell surface (121). The study also revealed that mAb43 binding to the cell-surface transporter could prevent autoantibodies from recognizing β-cells within the pancreatic islets (121). Not only do these findings have potential benefits in predicting the risk and/or early diagnosis of type 1 diabetes, the Fu lab has additionally demonstrated that mAb43 binds to extracellular loops of ZnT8 on the β-cell surface, masking its antigenic exposure and preserving in vivo insulin secretion against persistent insulitis (123). This provides a novel antigen-specific immunotherapeutic strategy to prevent and reverse clinical type 1 diabetes.

Conclusion

Although membrane proteins constitute more than 50% drug targets, transition metal transporters have rarely featured in this category. We postulate that the relative lack of understanding of the role of these proteins in controlling trace metal homeostasis is a major contributing factor. The paucity of in vitro reconstitution experiments, the challenge of redox active transition metal ions as substrates, and the substrate promiscuity of metal transporters have all contributed to the lack of comprehensive understanding of transition metal transporters. The difficulties of isolating and purifying integral membrane transporters present yet another major challenge. Significant advancements have been made over the past decade that will continue to strengthen our understanding of the mechanisms of transition metal transport and homeostasis. Further investigations of transition metal transporters, notably substrate identity, metal coordination chemistries, and interactions with other proteins will be necessary to understand the structural chemistry of these very important proteins. The ability to resolve the atomic resolution structure of more metal transporters, particularly with bound metal ions, will most likely uncover unknown features that in combination with in vitro reconstitution data, would delineate new transport mechanisms that will further enhance our understanding of the physiological regulation of essential trace metals. Nonetheless, current techniques in structural approaches still present some bottlenecks which range from size limitation in cryo-EM to high quantity of protein needed for crystallography studies. Therefore, complementary biochemical and biophysical experiments are necessary to better understand the role of transition metal transporters in human health and diseases.

Supporting information

This article contains supporting information.

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Supporting information

Supporting Figure

Acknowledgments

We acknowledge support from the intramural program of the 10.13039/100009633 Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) and the 10.13039/100000002 National Institutes of Health (NIH) (ZIAHD008928 ).

Author contributions

E. L. O. and A. B. conceptualization; E. L. O. and A. B. writing–original draft; E. L. O. and A. B. writing–review and editing; A. B. supervision; A. B. funding acquisition.

Funding and additional information

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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