Structures, Mechanisms, and Physiological Functions of Zinc Transporters in Different Biological Kingdoms

Abstract

Zinc transporters take up/release zinc ions (Zn²⁺) across biological membranes and maintain intracellular and intra-organellar Zn²⁺ homeostasis. Since this process requires a series of conformational changes in the transporters, detailed information about the structures of different reaction intermediates is required for a comprehensive understanding of their Zn²⁺ transport mechanisms. Recently, various Zn²⁺ transport systems have been identified in bacteria, yeasts, plants, and humans. Based on structural analyses of human ZnT7, human ZnT8, and bacterial YiiP, we propose updated models explaining their mechanisms of action to ensure efficient Zn²⁺ transport. We place particular focus on the mechanistic roles of the histidine-rich loop shared by several zinc transporters, which facilitates Zn²⁺ recruitment to the transmembrane Zn²⁺-binding site. This review provides an extensive overview of the structures, mechanisms, and physiological functions of zinc transporters in different biological kingdoms.

1. Introduction

Zinc ions (Zn²⁺), an essential trace element in bacteria, fungi, plants, and animals, including humans [1], serve as a key component in many signal transduction processes and act as an essential cofactor for many proteins and enzymes [2,3]. Zinc deficiency causes several human diseases [4,5,6,7,8,9,10,11,12,13]; indeed, zinc supplements have beneficial effects on human health [8,14,15,16,17,18,19]. However, excessive adsorption of Zn²⁺ leads to disruption of the gastrointestinal flora balance, deficiency of other essential heavy metals, including iron, copper, and manganese, and reduction in immune function [20,21,22,23]. Zn²⁺ also plays an important role in the physiology of organisms such as plants and bacteria [24,25]. In plants, zinc deficiency is linked to growth defects and inhibition of flowering [26,27]. Additionally, Zn²⁺ is responsible for the virulence of some bacteria [28]. Since Zn²⁺ is involved in numerous biological events, humans, plants, yeasts, and bacteria have evolved elaborate Zn²⁺ transport systems that respond to Zn²⁺ perturbation.

Failure of the Zn²⁺ transport systems plays a role in diseases such as cancer [29,30], Alzheimer’s [31,32], and Parkinson’s [33,34], as well as temporary zinc deficiency in newborns [35], perinatal fatal cardiomyopathy [36], risk of febrile seizures [37], Lowe’s syndrome [38], disorders of muscle tone with polycythemia [39,40], and chronic liver disease [40]. Therefore, human zinc transporters (ZnTs) are potential targets of drugs and preclinical diagnostic tests. Owing to the important physiological roles, and pharmacological and preclinical diagnostic significance of Zn²⁺ transport systems, a variety of biochemical, structural, physiological, and genetic experiments have been carried out over the past several decades to better understand their functions and mechanisms. The most comprehensively studied bacterial zinc transporter is YiiP, which works in Escherichia coli and Shewanella oneidensis (EcYiiP and SoYiiP, respectively) [41,42,43,44,45,46,47,48,49]. These transporters are a convenient model to study the general mechanisms underlying Zn²⁺ transport. The most intensively studied mammalian ZnTs are SLC30A7/ZnT7 [50] and SLC30A8/ZnT8 [51,52]. Our interests in ZnT family members stem mainly from their roles in maintaining Zn²⁺ homeostasis in cellular organelles throughout the body and the fact that their dysfunction causes serious diseases.

As is the case for other membrane transporters, ZnTs undergo conformational conversion to transport Zn²⁺ across biological membranes. To fully understand the mechanism underlying Zn²⁺ transport, high-resolution structures of the transporters have been captured in different states. The first X-ray crystal structure of a zinc transporter (

Table 1. X-ray and cryo-EM structure of zinc transporters (ZnTs).

Proteins	Main Functions	Organisms	States	Conformations (PDB Code)	Ligands	Methods	References
YiiP	Transport Zn2+ out of the cytoplasm and into the periplasm	Escherichia coli	Homodimer	Outward-facing (2QFI, 3H90)	Zn2+	X-ray diffraction	[41,42]
		Shewanella oneidensis	Homodimer	Inward-facing (3J1Z, 5VRF, 7KZZ (1))	Zn2+	Electron microscopy	[44,45,46]
			Homodimer	Inward-facing occluded (7KZX)	Zn2+		[43]
ZnT7	Transport Zn2+ out of the cytoplasm and into the Golgi lumen	Homo sapiens	Homodimer	Outward-facing (8J7T)	Apo	Electron microscopy	[50]
			Homodimer	Outward-facing (8J7U)	Zn2+
			Heterodimer	Inward-facing and outward-facing (8J7V (2))	Apo
			Heterodimer	Inward-facing with Zn2+ and outward-facing (8J80 (3))	Zn2+, Apo
			Heterodimer	Inward-facing with Zn2+ and outward-facing with Zn2+ (8J7W) (4)	Zn2+
ZnT8	Transport Zn2+ out of the cytoplasm and into the insulin secretory granule	H. sapiens	Homodimer	Outward-facing (6XPE)	Zn2+	Electron microscopy	[51]
			Heterodimer	Outward-facing and inward-facing (6XPF)	Apo
		Xenopus tropicalis	Homodimer	Outward-facing (7Y5G)	Zn2+		[52]
			Homodimer	Outward-facing (7Y5H (5))	Apo

⁽¹⁾ This structure was observed in the presence of 0.5 mM EDTA. ⁽²⁾ This structure was observed in the absence of Zn²⁺. ⁽³⁾ This structure was observed in the presence of 10 μM Zn²⁺. ⁽⁴⁾ This structure was observed with addition of 200 and 300 μM Zn²⁺. ⁽⁵⁾ This structure was observed at low pH.

) was reported for EcYiiP [41,42], followed by the EM structure of SoYiiP [43,44,45,46]. More recently, cryo-EM structures of vertebrate ZnTs have been reported (Table 1); these include Homo sapiens ZnT7 (HsZnT7) [50], Homo sapiens ZnT8 (HsZnT8) [51], and Xenopus tropicalis ZnT8 (XtZnT8) [52]. These structures allow us to propose an updated model of ZnTs-mediated Zn²⁺ transport. Of note, our recent structural and biochemical studies on HsZnT7 revealed the role of its cytosolic histidine-rich loop (His-loop) in efficient Zn²⁺ uptake [50]. Thus, we have built on the structural and mechanistic foundations of ZnTs in the biological kingdom, while making significant progress regarding research into other members with Zn²⁺ transport functions.

2. Zn²⁺ Transport Systems in Prokaryotes and Eukaryotes

Prokaryotes and eukaryotes have developed a variety of Zn²⁺ transport systems to promote the uptake or efflux of Zn²⁺ across biological membranes. ZnTs can be divided into three major groups depending on the mode of transport: Uniporters that transport Zn²⁺ alone; symporters that transport Zn²⁺ in the same direction as other ions, such as protons; and antiporters that transport Zn²⁺ and another ion in opposite directions, such that the binding of one is concomitant with the release of the other. In general, uniporters require no external energy input and transport specific molecules along their concentration gradients; they are therefore passive transporters. However, it can also act as an active transporter if the transport process is against the concentration gradient. By contrast, symporters and antiporters use the energy stored in the concentration gradient of another ion, in many cases, a proton, to transport specific molecules against their concentration gradients. In this regard, symporters and antiporters can be regarded as active transporters. In addition, some P-ATPases and ABC transporters transport Zn²⁺ using ATP as an external energy source to overcome the Zn²⁺ concentration gradient.

Zinc transporters (ZnTs) and ZRT- and IRT-related proteins (ZIPs) are the two major Zn²⁺ transport families found universally in bacteria, yeasts, plants, and animals, including humans. ZnTs and ZIPs selectively transport Zn²⁺, but in opposite directions: ZnTs export Zn²⁺ from the cytoplasm, whereas ZIPs import Zn²⁺ into the cytoplasm. Thus, ZnTs and ZIPs play important roles in maintaining homeostasis of intracellular and intra-organelle Zn²⁺ levels.

While ZntB from Escherichia coli (EcZntB) acts as a Zn²⁺/H⁺ symporter [53], many ZnTs function as proton-driven antiporters, exchanging H⁺ in the extracellular space or organelle lumens for Zn²⁺ in the cytoplasm [41,42,43,44,45,46,47,48,49,50,51,52,53,54,55]. By contrast, there is no clear evidence that ZIPs use proton energy flux to transport Zn²⁺ across the membranes. However, recent biochemical studies suggest that, like ZnTs, Bordetella bronchiseptica ZIP (BbZIP) may function as a Zn²⁺/H⁺ antiporter [56].

3. ZnTs

ZnTs belong to the cation diffusion facilitator (CDF) family, which can be classified into three groups: Zn-CDFs, Zn/Fe-CDFs, and Mn-CDFs [57,58]. Zn-CDFs consist of Zn²⁺ and Co²⁺ transporters, including ZitB-like, ZnT1-like, and Zrc1-like proteins. The ZitB-like clusters are from E. coli. The ZnT1-like clusters include only metazoans. The Zrc1-like cluster includes only fungal CDFs originating from Ascomycetes, Basidiomycetes, and Zygomycetes. Zn/Fe-CDFs are cation-efflux pumps that transport Fe²⁺ or Zn²⁺, and also Co²⁺, Cd²⁺, and Ni²⁺. Mn-CDFs include metal tolerance proteins (MTPs) from plants.

3.1. Mammalian ZnTs

Ten ZnTs (ZnTs 1–10) have been identified in mammals, including humans [59,60]. All ZnTs are Zn-CDF members, although ZnT10 is more likely a manganese transporter [59,60,61]. Based on their amino acid sequence similarities, ZnTs are divided into four subgroups: Group 1 includes ZnT5 and ZnT7; group 2 includes ZnT2-ZnT4 and ZnT8; group 3 includes ZnT1 and ZnT10; and group 4 includes ZnT6 and ZnT9 [60]. Most ZnTs form a homodimer composed of the same protomers [50,51,52], whereas ZnT5 and ZnT6 form a heterodimer including two different protomers [62], and all are located on the plasma or organelle membranes, where they control intracellular and extracellular Zn²⁺ balance [59,63]. Specifically, ZnT7 transports Zn²⁺ into the lumen of the pre-cis- and cis-Golgi, whereas ZnT5/6 and ZnT4 transport Zn²⁺ into the lumen of the medial- and trans-Golgi [64]. ZnT7 and ZnT5/6 are responsible for the Golgi-to-ER retrograde transport of the ER chaperone ERp44 [64]. This system is involved in the maturation and activation of some secretory proteins during transport through the early secretory pathway [65].

3.2. Plant ZnTs

Metal tolerance proteins (MTPs) are bivalent cationic transporters in plants that play crucial roles in metal tolerance and homeostasis in metal non-hyperaccumulators (e.g., Arabidopsis thaliana) and hyperaccumulators (e.g., Arabidopsis halleri and Noccaea caerulescens) [66]. MTPs are classified into seven groups based on their amino acid sequence similarities [67]. Thus, plant MTPs are very diverse so as to satisfy the need to absorb or detoxify specific metals. A. thalaina has 12 MTPs, while P. trichocarpa MTP has up to 22 MTP genes [68]. In A. thaliana, AtMTP1 and AtMTP3 ZnTs localized on the vacuole membrane maintain Zn²⁺ homeostasis [69,70,71]. AtMTP1 and AtMTP3 are involved in the sequestration of excess cytoplasmic Zn²⁺ into vacuoles [71]. Whereas AtMTP1 is more ubiquitously expressed, expression of AtMTP3 is restricted to the root epidermis and cortex [69,72]. Like mammalian ZnT5 and ZnT6, AtMTP5 and AtMTP12 form a heterodimer at the Golgi membrane and transport Zn²⁺ into the Golgi lumen [73].

3.3. Yeast ZnTs

Our understanding of ZnTs in yeast derives primarily from Saccharomyces cerevisiae. In S. cerevisiae, vacuolar ZnTs ZRC1 and COT1 act as Zn²⁺/H⁺ antiporters and regulate Zn²⁺ homeostasis by transporting and storing Zn²⁺ in the vacuole [74,75]. ScZRC1 senses Zn²⁺ availability in the cytosol, possibly through the histidine-repeat motifs, and transports Zn²⁺ from the cytosol to the vacuole when cytosolic Zn²⁺ is abundant, thereby conferring resistance to Zn²⁺ toxicity [76,77].

S. cerevisiae also possesses Msc2 and Zrg17, which transport Zn²⁺ from the nucleus and ER to the cytoplasm [78]. ScMsc2 and ScZrg17 interact physically to form a heterodimer and likely serve to maintain the Zn²⁺ levels in the ER of Zn²⁺-adequate cells [79,80,81]. Schizosaccharomyces pombe also has a zinc transporter, called ZHF1, which maintains Zn²⁺ homeostasis in the ER and nucleus and sequesters Cd²⁺ into the ER [82]. The structures of yeast ZnTs have not yet been reported. While ScZRC1, ScCOT1, and ScZrg17 are predicted to have six transmembrane (TM) helices, ScMsc2 is presumed to contain up to 16 TM helices.

3.4. Bacterial ZnTs

Bacterial ZnTs YiiP, ZitB, and CzcD have been functionally characterized. Insight into the structural features and Zn²⁺ transport mechanisms of bacterial ZnTs comes primarily from YiiP. YiiP was first identified in Escherichia coli [83]. In vitro, YiiP also binds Hg²⁺, Co²⁺, Ni²⁺, Mn²⁺, Ca²⁺, and Mg²⁺ but is unlikely to transport them efficiently [84]. Like mammalian ZnTs, YiiP functions as a Zn²⁺/H⁺ antiporter [43,48].

Other ZnTs have been identified recently in bacteria. ZitB conducts Zn²⁺ efflux across the cytoplasmic membrane, thereby reducing Zn²⁺ accumulation in the cytoplasm and rendering bacteria more resistant to Zn²⁺ [85]. By contrast, ZntA, a Zn²⁺-transporting P-ATPase, is required for growth at more toxic concentrations [85]. CzcD is a Cd²⁺, Co²⁺, and Zn²⁺/H⁺-K⁺ antiporter involved in maintaining intracellular divalent cation and potassium homeostasis through active efflux of Zn²⁺, Cd²⁺, and Co²⁺ in exchange for K⁺ and protons [86].

4. Structural Basis of ZnTs

In general, ZnTs adopt inward-facing (IF) or outward-facing (OF) forms and undergo conversion between these two forms during Zn²⁺ transport (Figure 1A). The IF form creates an open cavity for Zn²⁺ recruitment from the cytoplasm to the TM metal-binding site. Conversely, in the OF form, an open cavity is formed on the extracellular or organelle luminal side to release Zn²⁺ (Figure 1A). Unlike ZnTs, ZIPs are supposed to operate by a mechanism named elevator-type transport. Membrane transporters using this mechanism commonly consist of moving and fixed domains. Switching between the outward- and inward-facing forms involves the sliding of the entire moving domain through the bilayer as a rigid body. The substrate-binding site translocates some distance across the bilayer during transport, along with the moving domain [87].

The first X-ray crystal structure of ZnTs was solved for EcYiiP in a Zn²⁺-bound OF state [41,42]. Subsequently, cryo-EM single-particle analysis identified the structure of a Zn²⁺-bound IF state for SoYiiP [44,46] (Table 1). More recently, the cryo-EM structures of vertebrate ZnTs human ZnT7 (HsZnT7) [50], human ZnT8 (HsZnT8) [51], and Xenopus ZnT8 (XtZnT8) [52] were reported (Table 1), revealing that these ZnTs can adopt both OF-OF homodimeric and IF-OF heterodimeric conformations [50,51,52]. The presence of the homodimeric and heterodimeric conformations may suggest that the two protomers work independently during Zn²⁺ transport [50,51,52], although more detailed studies are required to draw a firm conclusion.

All Zn-CDFs share common structural folds. The structural core consists of six TM helices (TM1-TM6) in the TM domain (TMD), and a cytosolic domain (CTD) with a ferredoxin-like fold having an αββαβ secondary structure topology (Figure 1B). The Zn²⁺-binding site is formed by an HXXXD motif located on TM2 and TM5, individually, near the center of the TMD (Figure 1C). In some Zn-CDFs, the His (H) and Asp (D) residues on the motifs are replaced by Asp (D) and Asn (N), respectively (Figure 1D). Some eukaryotic Zn-CDFs possess a histidine-rich loop (His-loop) flanked by TM4 and TM5. The length of the His loop, and the number and distribution of His residues in the loop, vary among the Zn-CDFs (Figure 2).

As aforementioned, all ZnTs belong to the SLCA30 family, allowing us to compare the conformational details between them. ZnTs show different TM helix arrangements in both the IF and OF forms. In the IF form of bacterial YiiP and HsZnT8, TM4 and TM5 largely swing to the outside on the cytosolic side (relative to their positions in the OF form), using their luminal ends as pivot points (Figure 3A,B). By contrast, TM2 moves slightly toward the center [51]. Notably, in the IF form of HsZnT7, TM5 kinks at the middle, and its cytosolic half is largely bent toward the outside, resulting in the very open cytosolic cavity (Figure 3C) [50]. In this form, part of the His loop is integrated into the cytosolic cavity, and His164 in this loop is directly coordinated to Zn²⁺ along with His70, Asp74, and Asp244 (Figure 3D) [50]. The detailed mechanistic and functional roles of the His-loop are discussed later.

There are also significant differences in the OF forms of ZnTs with a known structure. The OF form of HsZnT7 has a wider cavity at the luminal side than that of HsZnT8 and EcYiiP due to the more “outside” positions of TM1 and TM2 (Figure 3E,F). An additional conformation has been identified for Zn²⁺-unbound HsZnT7, in which TM5 packs tightly with TM2, TM3, and TM6 on both the cytoplasmic and luminal sides (Figure 3G) [50]. The TM helix arrangement in this state is highly superimposable to that of a previously reported occluded state of Zn²⁺-unbound SoYiiP (Figure 3G) [44]. Although the physiological relevance of the Zn²⁺-unbound occluded state remains unclear, this state may be formed after the release of Zn²⁺ to the luminal or periplasmic side and before complete conversion to the IF state with a widely open cytosolic cavity. Hereafter, we refer to this state as the “IF resting state” in this review article.

5. Zn²⁺-Binding Sites on ZnTs

Zn²⁺-Binding Sites and Metal Ion Selectivity

Metal specificity is an important functional feature of all metal transporters. X-ray crystallographic and cryo-EM analyses revealed one to three Zn²⁺-binding sites (site A, site B, and site C) in ZnTs. All ZnTs commonly possess a Zn²⁺-binding site in the TMD (site A). Zn²⁺ binds transiently to site A before effluxing to the other side, indicating that site A is located on the Zn²⁺-translocation pathway. Site A contains highly conserved Zn²⁺-binding motifs formed by the BXXXB motif on TM2 and TM5, in which B is His (H), Asp (D), Asn (N), or Glu (E), and X can be any residues (Figure 1C,D). There are, however, some differences in the sequence of site A (Figure 1D). While human, plant, and yeast ZnTs conserve the (HXXXD)^TM2-(HXXXD)^TM5 motif, different amino acid sequences are seen in the motifs of bacterial ZnTs; for instance, (DXXXD)^TM2-(HXXXD)^TM5 in EcYiiP and SoYiiP, and (HXXXD)^TM2-(HXXXD)^TM5 in EcZitB and CzcD (Figure 1D). Notably, mammalian ZnT6 loses the BXXXB motif in the TMD, and does not therefore have Zn²⁺ transport activity [62]. Mammalian ZnT10 harboring (NXXXD)^TM2-(HXXXD)^TM5 transports Mn²⁺ rather than Zn²⁺ (Figure 1D) [88,89,90]. AtMTP11 and ShMTP8 harbor (DXXXD)^TM2-(DXXXN)^TM5 and transport both Mn²⁺ and Cu²⁺ (Figure 1D) [66]. Thus, not all ZnTs transport Zn²⁺ exclusively.

Site B is less conserved at the TMD-CTD interface (Figure 1B and Figure 4A). In bacterial YiiP, this site is located in the TM2-TM3 loop, which contains a DHH motif (Figure 4B) [42,43,44]. In HsZnT8, this site is constituted by His residues from the TM2-TM3 loop in the TMD and from the α2-β3 loop in the CTD, and it has low affinity for Zn²⁺ [51]. While the functional role of site B is unclear in HsZnT8, bacterial YiiP participates directly in Zn²⁺ transport [43,44]. Since site B is positioned near the cytosolic Zn²⁺ entry gate, mutation of two His residues at site B reduces the Zn²⁺ transport activity of bacterial YiiP [43]; thus, site B is thought to trap Zn²⁺ in the cytosol, thereby facilitating Zn²⁺ transport [43,51]. Although the structure of AtMTP1 has not been solved, homology modeling based on the crystal structure of EcYiiP suggests that it lacks site B (Figure 4B). However, mutations of some residues in the TM2-TM3 loop impair the Zn²⁺ transport activity of AtMTP1, indicating that the TM2-TM3 loop itself is essential for AtMTP1 function [91].

Site C is located at the dimer interface between two CTDs (Figure 1B and Figure 4A). In bacterial YiiP, site C forms a binuclear Zn²⁺ complex composed of the (HHD)₂ motif and stabilizes its dimeric conformation (Figure 4C,D) [42,43]. In HsZnT8, the exact location of site C differs from that in bacterial YiiP. In HsZnT8, two Zn²⁺ ions are coordinated by an HCH motif from the N-terminal loop, and a Cys-Cys pair from the C-terminal tail, thereby forming a tetrahedral complex (Figure 4D) [51]. The HCH motifs seal off site C and bury the Zn²⁺ ions inside the protein (Figure 4D). This motif is highly conserved among the ZnT8 orthologues but not among bacterial ZnTs [51]. The N-terminal truncation that accompanies the loss of the HCH motif reduces the Zn²⁺ uptake activity of HsZnT8 significantly [51]. AtMTP1 also possesses site C within the CTD (Figure 4C), and the lack of this domain results in loss of function [91]. Thus, Zn²⁺-mediated dimerization via the CTD seems likely to be essential for Zn²⁺ transport by HsZnT8, bacterial YiiP, and plant MTP1.

By contrast, no Zn²⁺ ions have been identified at either site B or site C of HsZnT7, although its cryo-EM structures were determined in the presence of high Zn²⁺ concentrations (10, 200, or 300 μM) of Zn²⁺ (Figure 4). Consistent with this, residues required for Zn²⁺ binding are not conserved at site B or site C of HsZnT7 (Figure 4C). Presumably, other elements contribute to the dimerization of the CTD and the Zn²⁺ transport activity of HsZnT7. Indeed, dimerization of the CTD is mediated by residues within four β-strands and TM2-TM3 loops located at the dimer interface (Figure 4A) [50].

6. Mechanism of Zn²⁺ Transport by Human ZnT7 and Bacterial YiiP

6.1. Zn²⁺ Transport by HsZnT7

HsZnT7 transports Zn²⁺ from the cytoplasm to the TM Zn²⁺-binding site (site A), and then to the Golgi lumen. The cryo-EM structures of HsZnT7 in multiple states have helped to paint a full picture of Zn²⁺ transport mediated by this transporter (Figure 5) [50]. In the absence of Zn²⁺, TM5 packs against TM2, TM3, and TM6 at both the cytosolic and luminal sides, forming an “IF resting” state (Figure 3G and Figure 5A). In this state, Zn²⁺ uptake to site A seems to be blocked due to the closed Zn²⁺ entry gate (Figure 5A–C(i)). In the presence of Zn²⁺, however, the cytosolic cavity opens by bending the N-terminal half of TM5, and the His-loop is integrated into the cavity to coordinate with Zn²⁺ at site A (Figure 5A–C(ii)). In the next step, TM5 returns to a straight conformation concomitant with pulling His164 out of site A. Consequently, His240 on TM5 coordinates with Zn²⁺ instead of His164 (Figure 5A–C(iii)). Upon conversion to the OF state, the His70 side chain moves away from Zn²⁺ slightly, while Asp74, His240, and Asp244 remain coordinated with Zn²⁺ (Figure 5B(iv)). Simultaneously (or shortly thereafter), protons presumably enter the Zn²⁺ transport channel from the weakly acidic Golgi lumen to protonate His70 and His240. As a result, His70 flips out of site A, and Zn²⁺ is released to the Golgi lumen (Figure 5A–C(v)). In this context, it is notable that while a His residue is highly conserved among human ZnTs, it is substituted by an Asp residue in bacterial YiiP (Figure 1C,D). Although it is not fully elucidated how many protons are involved in the release of Zn²⁺, the above mechanistic model suggests that HsZnT7 exchanges one Zn²⁺ for two protons per cycle.

6.2. Zn²⁺ Transport by Bacterial YiiP

Recent biochemical, structural, and computational studies provide deep insight into the mechanism of Zn²⁺ transport mediated by bacterial YiiP [43,44]. Initially, Zn²⁺ is recruited to site B, formed by the TM2-TM3 loop, and then transferred to site A of another protomer (Figure 6) [43,44]. In the Zn²⁺-free IF state, the TM2-TM3 loop is disordered to allow Zn²⁺ to approach site B. After binding Zn²⁺ site B, the TM2-M3 loop forms an ordered conformation [43]. Importantly, the higher affinity of Zn²⁺ for site A than site B explains sequential Zn²⁺ transfer from site A to site B [43]. At this step, the negatively charged cytoplasmic cavity seems advantageous for directing Zn²⁺ to site A [43,92]. In the OF state, the TM2-TM3 loop in a Zn²⁺-free state interacts with the CTD, forming an extended conformation [43]. Zn²⁺ is released to the periplasm and is facilitated by the protonation of two residues in site A, or potentially three residues at a lower pH [43].

MD simulations using EcYiiP and SoYiiP as a model metal transporter demonstrate that Zn²⁺ binds transiently to Asp150 (Asp152 in SoYiiP) on TM5, and to Glu79 (Glu81 in SoYiiP) on TM3, before reaching site A (Figure 6) [92]. In the next step, His153 (His155 in SoYiiP), located at site A, plays a key role in guiding Zn²⁺ to site A. In support of this, His153 can adopt two conformations to receive Zn²⁺ from Asp150, after which it forms a tetrahedral Zn²⁺ complex with Asp45, Asp49, and Asp157 (Figure 6) [49]. Release of Zn²⁺ can be triggered by protonation of site A residues [43,44]. His153 is likely deprotonated again when it faces the neutral-pH cytoplasm to receive Zn²⁺ from site B via Asp150 [49,51,54,92,93].

6.3. Role of the Proton Gradient in ZnT-Mediated Zn²⁺ Transport

Lactose permease (LacY) utilizes a proton motive force for the efficient transport of lactose [94]. Presumably, ZnTs also uses the proton motive force to transport Zn²⁺ from the cytoplasm to the extracellular space or the organelle lumen [55]. Given that the concentration of labile Zn²⁺ in the Golgi is much higher than in the cytosol [64,95,96], and that the Golgi is a weakly acidic organelle [97], it makes sense that Golgi-resident ZnTs utilize the energy provided by the Golgi-to-cytosol proton flow to transport Zn²⁺ to the luminal side. As mentioned above, protonation of His70 and His240 is critical for the IF-to-OF conversion and subsequent release of Zn²⁺. While HsZnT7 exchanges one Zn²⁺ for (possibly) two H⁺ per cycle, EcZitB [47] and EcYiiP [48] exchange one Zn²⁺ for one H⁺. SoYiiP conducts the exchange at a ratio of 1:2–3, depending on the external pH [43]. Human ZnT2 is thought to operate at a ratio of 1:2 [54].

7. Structural and Functional Roles of the Histidine-Rich Loop (His-Loop)

The His-loop is a unique sequence located at the cytoplasmic side between TM4 and TM5 (Figure 1B and Figure 2). The length of this loop, as well as the number and arrangement of His residues, can differ (Figure 2). Most bacterial ZnTs (e.g., YiiP and MntE), several mammalian ZnTs (e.g., ZnTs 6, 9, and 10), and plant MTPs (e.g., AtMTPs 5, 6, and 11) possess a short TM4-TM5 loop without any His residues. Mammalian ZnTs such as ZnTs 2-4 and ZnT8 are also characterized by a short His-loop. Of note, several mammalian ZnTs (e.g., ZnTs 1, 5, and 7), plant MTP1s (e.g., AtMTP1, NgMTP1, OsMTP1, and HvMTP1), and yeast ZnTs (e.g., SpZHF1, ScZRC1, and ScCOT1) have an exceptionally long His-loop (Figure 2). Despite having a relatively long TM4-TM5 loop, ZnT10 has no His residues in the loop (Figure 2). Such variations in the His-loop may underlie the diverse functions and different mechanisms of Zn²⁺ transport mediated by ZnTs. Indeed, the His-loop also functions as a Zn²⁺-buffering pocket [98], a Zn²⁺ selectivity filter [99], a Zn²⁺ sensor [98,100,101], or a Zn²⁺-fishing loop [50].

As described above, cryo-EM analysis of HsZnT7 revealed that a short segment of the His-loop inserts into the cytoplasmic cavity, where His164 contained in the His-loop coordinates directly with Zn²⁺ at site A, while His240 on TM5 is kicked out from site A due to bending of the N-terminal half of TM5 (Figure 5A,B(ii)) [50]. Thus, the His-loop appears to contribute to efficient Zn²⁺ uptake to site A, but its task seems transient. In other words, HsZnT7 may use the His-loop as a “Zn²⁺ fishing loop”, and His residues in the loop function as a “fishing hook” that captures Zn²⁺ and transfers it to site A. Consistent with this, the deletion of the His-loop, or the mutations of all His residues in the loop to Ser, abolishes the Zn²⁺ transport activity of HsZnT7 [50]. Similarly, deletion of the entire His-loop segment in Noccaea caerulescens MTP1 and AtMTP1 results in substantial loss of Zn²⁺ transport activity [98,102] although deletion of the first half of the His-loop increases the activity of AtMTP1 significantly [98].

The 63-residue His-loop of HsZnT7 contains 21 His residues and can be divided into two halves: The first half contains seven His residues close to the C-terminus of TM4, and the second half contains 14 His residues close to the N-terminus of TM5 (Figure 2) [50]. The His-loop of HsZnT7 binds Zn²⁺ with a K_d value of 12 μM; although each half can bind one Zn²⁺, the first and second halves appear to bind Zn²⁺ co-operatively [50]. AtMTP1 also contains an exceptionally long His-loop containing 25 His residues and can also be divided into two halves: The first half contains 18 His residues close to the C-terminus of TM4, while the second half contains seven His residues close to the N-terminus of TM5 (Figure 2) [98]. The His-loop of AtMTP1 can bind four Zn²⁺ ions, with a K_d value of 25 μM [101]. The lack of either half leads to impairment of Zn²⁺-binding by AtMTP1, suggesting that the two halves of the His-loop work co-operatively to form multiple Zn²⁺-binding sites [101]. However, stable Zn²⁺ binding may lead to slower Zn²⁺ release from AtMTP1, which likely explains the less efficient Zn²⁺ transport by AtMTP1 WT than by the mutant lacking the first half [98,101]. Consistent with this, the His-loop of AtMTP1 adopts a β-strand conformation to suppress its Zn²⁺ transport activity at high concentrations of Zn²⁺, implying a role in sensing excessive Zn²⁺ levels in the cytosol [101].

8. Zn²⁺ Transport by Other Zn²⁺ Transport Systems

8.1. ZIPs

ZIPs play a crucial role in mediating metal import across the plasma membrane as well as export across organelle membranes. ZIPs are classified into four subfamilies based on phylogenetic relationships [102]. In humans, 14 ZIPs have been identified. Despite their relevance to human diseases, the structural and mechanistic details of mammalian ZIPs remain poorly understood. Recent studies on Bordetella bronchiseptica ZIP (BbZIP) have shed light on some structural and mechanistic features [56,103,104]. Crystal structures of BbZIP revealed an IF monomer, with binuclear metal-binding sites potentially occupied by Zn²⁺ or Cd²⁺ [104], while cryo-EM studies revealed an IF homodimer [56]. BbZIP is an elevator-type transporter in which a four-TM helix bundle with bound metal ions slides as a rigid body against the dimeric domain composed of the other static TM helices to exert an alternating access mechanism, and its Zn²⁺ uptake is pH-dependent [56,105,106,107]. However, it is unclear whether ZIPs transfer Zn²⁺ through a symport or antiport mechanism [56,105]. The presence of a His-rich loop in many mammalian ZIPs, including BbZIP, adds complexity to the mechanisms underlying their regulated Zn²⁺ transport [108,109]. Plants possess more ZIPs [110], but detailed structural and mechanistic characterization has not been reported for any of them.

8.2. ZntB

ZntB belongs to the prokaryotic CorA family [111,112]. E. coli and P. aeruginosa ZntB transport multiple metal ions, including Zn²⁺, Co²⁺, Ni²⁺, and Cd²⁺ [53,113]. ZntB forms homogeneous pentagonal structures that span the biological membrane, similar to other members of the CorA family. Each protomer consists of two TM helices and a large cytoplasmic domain [53,113]. ZnTB-mediated Zn²⁺ transport is stimulated by a pH gradient across the biological membrane. ZntB transports protons and Zn²⁺ together in the same direction, thereby working as a symporter [53]. In contrast to bacterial YiiP and human ZnTs, ZntB does not seem to use an alternating access mechanism mediated by conformational transitions between IF and OF forms [53]. The cryo-EM structure of PaZntB is similar to that of EcZntB and T. maritima CorA, but with different helical arrangements [53,113,114]. PaZntB is capable of transporting Zn²⁺, Mg²⁺, Cd²⁺, Ni²⁺, and Co²⁺. In contrast to other ZntBs, PaZntB is unlikely to use the proton motive force [53,113]. Thus, different mechanisms may operate among ZntBs from various bacterial species [53,111,113,115].

8.3. P-Type ATPases with Zn²⁺ Transport Activity

Zn²⁺-transporting P-type ATPases, which belong to class IB (P_IB-2-ATPases), are active zinc transporters. While no P-type ATPases with Zn²⁺ transport activity have been identified in mammals, several have been identified in bacteria and plants. In plants, the P_IB-2-ATPases, also known as the heavy metal ATPases (HMAs), are thought to play an important role in the transport of transition metals. In A. thaliana, AtHMA2 and AtHMA4 are zinc transporters that increase Zn²⁺ levels in roots, stems, and leaves [66,116]. AtHMA1 functions to detoxify Zn²⁺ in the chloroplast, while AtHMA3 is thought to supply Zn²⁺ to the ER and vacuole. Little is known about the structural features and Zn²⁺ transport mechanisms of HMAs [66]. In bacteria, ZntA is an active zinc transporter that is crucial for cellular detoxification and sub-cellular redistribution of Zn²⁺ [117]. ZntA couples ATP hydrolysis with Zn²⁺ transport via the “Post-Albers” cycle, during which at least four primary intermediate states (E1, E1P, E2P, and E2) accumulate [118,119,120,121]. Structurally, P_IB-2-ATPases possess four domains: A TMD, a nucleotide-binding domain (N domain), an actuator/dephosphorylation domain (A domain), and a phosphorylation domain (P domain) [118,120,122]. In addition, P_IB-2-ATPases contain metal-binding domains (MDBs) in the N-terminal region to promote metal recruitment to the TM metal-binding site (114, 116, 118). To date, only the crystal structure of Shigella sonnei ZntA has been reported [117], and the cryo-EM structures of other P_IB-2-ATPases are currently unknown.

8.4. Zn²⁺ Transport by ATP-Binding Cassette Transporters

ATP-binding cassette (ABC) transporters are a ubiquitous superfamily of integrated membrane proteins that transport various substrates across biological membranes by utilizing ATP as an external energy source [123]. In bacteria, ZnuABC is a specialist zinc transporter. It comprises three protein subunits: A periplasmic Zn²⁺-binding protein (ZnuA), an integrated membrane protein that transports Zn²⁺ across the cytoplasmic membrane (ZnuB), and an ATPase protein in charge of coupling Zn²⁺ transport to ATP hydrolysis (ZnuC) [124,125]. In most Gram-negative bacteria, ZnuABC expression is regulated by a Zur (zinc uptake regulatory) protein induced by Zn²⁺ acquisition [126]. The structure of ZnuABC and its mechanism of Zn²⁺ transport coupled to ATP hydrolysis are only poorly understood, although crystal structures have been solved for EcZnuA [124,127,128], and Salmonella enterica ZnuA [129].

9. Conclusions

As described above, while some members of the Zn-CDF family share structural and mechanistic features, there are considerable variations among them. It is widely accepted that ZnTs commonly operate as dimers; however, detailed structural analyses conducted so far show that whereas bacterial YiiPs adopt OF-OF and IF-IF homodimers, human ZnTs exist as either OF-OF homodimers or IF-OF heterodimers. No IF-IF homodimers have been observed for mammalian ZnTs, likely due to their conformational instability. Indeed, modes of the TM helix rearrangement between the IF and OF states among ZnTs seem to differ significantly. Of particular note, HsZnT7 undergoes marked bending of TM5 to allow efficient Zn²⁺ uptake. Concomitantly, a part of the long cytoplasmic His-loop is integrated into the negatively charged cytosolic cavity to facilitate acqusition and efficient transfer of Zn²⁺ to the TM Zn²⁺-binding site. In this state, His164 in the His-loop is coordinated directly with Zn²⁺ in the TMD. Subsequently, His240 (TM5) repaces His164, followed by the conversion from the IF to the OF state. In this context, the essential role of His164 needs to be further explored by conducting additional mutational and structural studies. It will also be interesting to see if another His residue in the His-loop can replace His164 for efficient Zn²⁺ recruitment when His164 is deleted. Recently, a de novo heterozygous variant of SLC30A7, His164Ser, was found in Joubert syndrome patients [130]. Although no SLC30A7 variants have yet been shown to cause human phenotypes or diseases, SLC30A7 is a candidate gene associated with Joubert syndrome [130].

Undoubtedly, multiple ZnTs work co-operatively to maintain Zn²⁺ homeostasis in cells. Indeed, disruption of Zn²⁺ homeostasis leads to disruption of protein homeostasis [64,131] and dysfunction of essential enzymes, transcription factors, and other biomolecules [132,133,134,135], eventually causing many fatal diseases [29,30,31,32,33,34,35,36,37,38,39,40]. In this regard, the mechanisms of Zn²⁺ homeostasis mediated by various kinds of ZnTs need to be understood more comprehensively. Zinc biology is an important field of research that encompasses biochemical, structural, computational, physiological, and medical approaches.