Cell-Surface ZnT8 Antibody Prevents and Reverses Autoimmune Diabetes in Mice.

Type 1 diabetes (T1D) is an autoimmune disease in which pathogenic lymphocytes target autoantigens expressed in pancreatic islets, leading to the destruction of insulin-producing ß-cells. Zinc transporter 8 (ZnT8) is a major autoantigen abundantly present on the ß-cell surface. This unique molecular target offers the potential to shield ß-cells against autoimmune attacks in T1D. Our previous work showed that a monoclonal antibody (mAb43) against cell-surface ZnT8 could home in on pancreatic islets and prevent autoantibodies from recognizing ß-cells. This study demonstrates that mAb43 binds to exocytotic sites on the ß-cell surface, masking the antigenic exposure of ZnT8 and insulin after glucose-stimulated insulin secretion. In vivo administration of mAb43 to NOD mice selectively increased the proportion of regulatory T cells in the islet, resulting in complete and sustained protection against T1D onset as well as reversal of new-onset diabetes. The mAb43-induced self-tolerance was reversible after treatment cessation, and no adverse effects were exhibited during long-term monitoring. Our findings suggest that mAb43 masking of the antigenic exposure of ß-cells suppresses the immunological cascade from B-cell antigen presentation to T cell-mediated ß-cell destruction, providing a novel islet-targeted and antigen-specific immunotherapy to prevent and reverse clinical T1D.

Cell-Surface Autoantibody Targets Zinc Transporter-8 (ZnT8) for In Vivo ß-Cell Imaging and Islet-Specific Therapies.

Type 1 diabetes (T1D) is a disease in which autoimmune attacks are directed at the insulin-producing ß-cell in the pancreatic islet. Autoantigens on the ß-cell surface membrane are specific markers for molecular recognition and targets for engagement by autoreactive B lymphocytes, which produce islet cell surface autoantibody (ICSA) upon activation. We report the cloning of an ICSA (mAb43) that recognizes a major T1D autoantigen, ZnT8, with a subnanomolar binding affinity and conformation specificity. We demonstrate that cell-surface binding of mAb43 protects the extracellular epitope of ZnT8 against immunolabeling by serum ICSA from a patient with T1D. Furthermore, mAb43 exhibits in vitro and ex vivo specificity for islet cells, mirroring the exquisite specificity of islet autoimmunity in T1D. Systemic administration of mAb43 yields a pancreas-specific biodistribution in mice and islet homing of an mAb43-linked imaging payload through the pancreatic vasculature, thereby validating the in vivo specificity of mAb43. Identifying ZnT8 as a major antigenic target of ICSA allows for research into the molecular recognition and engagement of autoreactive B cells in the chronic phase of T1D progression. The in vivo islet specificity of mAb43 could be further exploited to develop in vivo imaging and islet-specific immunotherapies.

Novel autoantibodies to the ß-cell surface epitopes of ZnT8 in patients progressing to type-1 diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by autoimmune destruction of insulin-producing ß-cells in pancreatic islets. Seroconversions to islet autoantibodies (IAbs) precede the disease onset by many years, but the role of humoral autoimmunity in the disease initiation and progression are unclear. In the present study, we identified a new IAb directed to the extracellular epitopes of ZnT8 (ZnT8ec) in newly diagnosed patients with T1D, and demonstrated immunofluorescence staining of the surface of human ß-cells by autoantibodies to ZnT8ec (ZnT8ecA). With the assay specificity set on 99th percentile of 336 healthy controls, the ZnT8ecA positivity rate was 23.6% (74/313) in patients with T1D. Moreover, 30 children in a longitudinal follow up of clinical T1D development were selected for sequential expression of four major IAbs (IAA, GADA, IA-2A and ZnT8icA). Among them, 10 children were ZnT8ecA positive. Remarkably, ZnT8ecA was the earliest IAb to appear in all 10 children. The identification of ZnT8ec as a cell surface target of humoral autoimmunity in the earliest phase of IAb responses opens a new avenue of investigation into the role of IAbs in the development of ß-cell autoimmunity.

Zinc transporters and their functional integration in mammalian cells.

Zinc is a ubiquitous biological metal in all living organisms. The spatiotemporal zinc dynamics in cells provide crucial cellular signaling opportunities, but also challenges for intracellular zinc homeostasis with broad disease implications. Zinc transporters play a central role in regulating cellular zinc balance and subcellular zinc distributions. The discoveries of two complementary families of mammalian zinc transporters (ZnTs and ZIPs) in the mid-1990s spurred much speculation on their metal selectivity and cellular functions. After two decades of research, we have arrived at a biochemical description of zinc transport. However, in vitro functions are fundamentally different from those in living cells, where mammalian zinc transporters are directed to specific subcellular locations, engaged in dedicated macromolecular machineries, and connected with diverse cellular processes. Hence, the molecular functions of individual zinc transporters are reshaped and deeply integrated in cells to promote the utilization of zinc chemistry to perform enzymatic reactions, tune cellular responsiveness to pathophysiologic signals, and safeguard cellular homeostasis. At present, the underlying mechanisms driving the functional integration of mammalian zinc transporters are largely unknown. This knowledge gap has motivated a shift of the research focus from in vitro studies of purified zinc transporters to in cell studies of mammalian zinc transporters in the context of their subcellular locations and protein interactions. In this review, we will outline how knowledge of zinc transporters has been accumulated from in-test-tube to in-cell studies, highlighting new insights and paradigm shifts in our understanding of the molecular and cellular basis of mammalian zinc transporter functions.

Down-regulation of the islet-specific zinc transporter-8 (ZnT8) protects human insulinoma cells against inflammatory stress.

Zinc transporter-8 (ZnT8) primarily functions as a zinc-sequestrating transporter in the insulin-secretory granules (ISGs) of pancreatic ß-cells. Loss-of-function mutations in ZnT8 are associated with protection against type-2 diabetes (T2D), but the protective mechanism is unclear. Here, we developed an in-cell ZnT8 assay to track endogenous ZnT8 responses to metabolic and inflammatory stresses applied to human insulinoma EndoC-ßH1 cells. Unexpectedly, high glucose and free fatty acids did not alter cellular ZnT8 levels, but proinflammatory cytokines acutely, reversibly, and gradually down-regulated ZnT8. Approximately 50% of the cellular ZnT8 was localized to the endoplasmic reticulum (ER), which was the primary target of the cytokine-mediated ZnT8 down-regulation. Transcriptome profiling of cytokine-exposed ß-cells revealed an adaptive unfolded protein response (UPR) including a marked immunoproteasome activation that coordinately degraded ZnT8 and insulin over a 1,000-fold cytokine concentration range. RNAi-mediated ZnT8 knockdown protected cells against cytokine cytotoxicity, whereas inhibiting immunoproteasomes blocked cytokine-induced ZnT8 degradation and triggered a transition of the adaptive UPR to cell apoptosis. Hence, cytokine-induced down-regulation of the ER ZnT8 level promotes adaptive UPR, acting as a protective mechanism that decongests the ER burden of ZnT8 to protect ß-cells from proapoptotic UPR during chronic low-grade inflammation.

Water molecules mediate zinc mobility in the bacterial zinc diffusion channel ZIPB.

Regulated ion diffusion across biological membranes is vital for cell function. In a nanoscale ion channel, the active role of discrete water molecules in modulating hydrodynamic behaviors of individual ions is poorly understood because of the technical challenge of tracking water molecules through the channel. Here we report the results of a hydroxyl radical footprinting analysis of the zinc-selective channel ZIPB from the Gram-negative bacterium, Bordetella bronchiseptica Irradiating ZIPB by microsecond X-ray pulses activated water molecules to form covalent hydroxyl radical adducts at nearby residues, which were identified by bottom-up proteomics to detect residues that interact either with zinc or water in response to zinc binding. We found a series of residues exhibiting reciprocal changes in water accessibility attributed to alternating zinc and water binding. Mapping these residues to the previously reported crystal structure of ZIPB, we identified a water-reactive pathway that superimposed on a zinc translocation pathway consisting of two binuclear metal centers and an interim zinc-binding site. The cotranslocation of zinc and water suggested that pore-lining residues undergo a mode switch between zinc coordination and water binding to confer zinc mobility. The unprecedented details of water-mediated zinc transport identified here highlight an essential role of solvated waters in driving zinc coordination dynamics and transmembrane crossing.

Induction of the metal transporter ZIP8 by interferon gamma in intestinal epithelial cells: Potential role of metal dyshomeostasis in Crohn's disease.

Transition metals are required for intestinal homeostasis and provide essential nutrients for the resident microbiota. Abnormalities in metal homeostasis are common in Crohn's disease (CD), but remain poorly defined and causes appear multifactorial. There has been renewed interest in understanding these mechanisms with the discovery of an association between a coding variant in SLC39A8 (rs13107325; ZIP8 A391T) and increased CD risk. SLC39A8 encodes the protein ZIP8, a metal transporter that is induced under inflammatory stimuli; however, studies of its gut-specific functions are lacking. Here, we show that SLC39A8 mRNA is differentially expressed in active CD with a high positive correlation with markers of disease severity, including CXCL8, TNFα, IFNγ, and calprotectin. SLC39A8 expression exhibits a negative correlation with SLC39A4 and SLC39A5, two key zinc importers in absorptive enterocytes, and a lack of correlation with two manganese transporters, SLC39A14 and SLC11A2. Immunohistochemistry demonstrates ZIP8 expression in intestinal epithelial cells and immune cells of the lamina propria. Patients with CD exhibit variable patterns of ZIP8 subcellular localization within IECs. In ileal enteroids, SLC39A8 was induced by IFNγ and IFNγ + TNFα, but not by TNFα alone, independent of NF-κB activation. IFNγ also down-regulated SLC39A5. To explore the functional implications of disease-associated genetic variation, in over-expression experiments in HEK293A cells, ZIP8 A391T was associated with increased TNFα-induced NF-κB activation, consistent with a loss of negative regulation. Taken together, these results suggest a potential role for ZIP8 in intestinal inflammation, induced by IFNγ in the intestinal epithelial compartment, and that perturbations in negative regulation of NF-κB by ZIP8 A391T may contribute to CD pathogenesis.

Highly specific monoclonal antibodies for allosteric inhibition and immunodetection of the human pancreatic zinc transporter ZnT8.

Solute carrier family 30 member 8 (SLC30A8), encoding the pancreatic zinc transporter ZnT8, is a susceptibility gene for type 2 diabetes (T2D). Reducing ZnT8 transport activity or down-regulating its cellular expression is hypothesized to be an antidiabetogenic strategy mimicking the protective effect of SLC30A8 haploinsufficiency in humans. However, research tools to inhibit ZnT8 activity and measure cellular ZnT8 levels are not available. Here, we report the identification of two anti-ZnT8 mAbs applicable to addressing these unmet needs. Both mAbs exhibited subnanomolar affinities for human ZnT8 and were selective against homologous zinc transporters with distinct cross-species reactivities and epitope recognition. We showed that antigen-binding fragments (Fabs) protected ZnT8 from unfolding and inhibited ZnT8-mediated zinc transport in proteoliposomes. Negative-stain EM revealed a ternary binding complex of a ZnT8 monomer and two different Fabs at a 1:1:1 stoichiometry. Moreover, dual bindings of two different mAbs to a single ZnT8 protein multiplied the individual anti-ZnT8 specificities, enabling quantification of cellular ZnT8 levels by homogeneous time-resolved fluorescence (HTRF). Our results demonstrate the utilities of the two generated mAbs as allosteric inhibitors and highly specific biosensors of human ZnT8.

A subclass of serum anti-ZnT8 antibodies directed to the surface of live pancreatic ß-cells.

The islet-specific zinc transporter ZnT8 is a major self-antigen found in insulin granules of pancreatic ß-cells. Frequent insulin secretion exposes ZnT8 to the cell surface, but the humoral antigenicity of the surface-displayed ZnT8 remains unknown. Here we show that a membrane-embedded human ZnT8 antigen triggered a vigorous immune response in ZnT8 knock-out mice. Approximately 50% of serum immunoreactivities toward ZnT8 were mapped to its transmembrane domain that is accessible to extracellular ZnT8 antibody (ZnT8A). ZnT8A binding was detected on live rat insulinoma INS-1E cells, and the binding specificity was validated by a CRISPR/Cas9 mediated ZnT8 knock-out. Applying established ZnT8A assays to purified serum antibodies from patients with type 1 diabetes, we detected human ZnT8A bound to live INS-1E cells, whereas a ZnT8 knock-out specifically reduced the surface binding. Our results demonstrate that ZnT8 is a cell surface self-antigen, raising the possibility of a direct involvement in antibody-mediated ß-cell dysfunction and cytotoxicity.

Proteoliposome-based full-length ZnT8 self-antigen for type 1 diabetes diagnosis on a plasmonic platform.

Identified as a major biomarker for type 1 diabetes (T1D) diagnosis, zinc transporter 8 autoantibody (ZnT8A) has shown promise for staging disease risk and disease diagnosis. However, existing assays for ZnT8 autoantibody (ZnT8A) are limited to detection by soluble domains of ZnT8, owing to difficulties in maintaining proper folding of a full-length ZnT8 protein outside its native membrane environment. Through a combined bioengineering and nanotechnology approach, we have developed a proteoliposome-based full-length ZnT8 self-antigen (full-length ZnT8 proteoliposomes; PLR-ZnT8) for efficient detection of ZnT8A on a plasmonic gold chip (pGOLD). The protective lipid matrix of proteoliposomes improved the proper folding and structural stability of full-length ZnT8, helping PLR-ZnT8 immobilized on pGOLD (PLR-ZnT8/pGOLD) achieve high-affinity capture of ZnT8A from T1D sera. Our PLR-ZnT8/pGOLD exhibited efficient ZnT8A detection for T1D diagnosis with ∼76% sensitivity and ∼97% specificity (n = 307), superior to assays based on detergent-solubilized full-length ZnT8 and the C-terminal domain of ZnT8. Multiplexed assays using pGOLD were also developed for simultaneous detection of ZnT8A, islet antigen 2 autoantibody, and glutamic acid decarboxylase autoantibody for diagnosing T1D.

Coupling of Insulin Secretion and Display of a Granule-resident Zinc Transporter ZnT8 on the Surface of Pancreatic Beta Cells.

The islet-specific zinc transporter ZnT8 mediates zinc enrichment in the insulin secretory granules of the pancreatic beta cell. This granular zinc transporter is also a major self-antigen found in type 1 diabetes patients. It is not clear whether ZnT8 can be displayed on the cell surface and how insulin secretion may regulate the level of ZnT8 exposure to extracellular immune surveillance. Here we report specific antibody binding to the extracellular surface of rat insulinoma INS-1E cells that stably expressed a tagged human zinc transporter ZnT8. Flow cytometry analysis after fluorescent antibody labeling revealed strong correlations among the levels of ZnT8 expression, its display on the cell surface, and glucose-stimulated insulin secretion (GSIS). Glucose stimulation increased the surface display of endogenous ZnT8 from a basal level to 32.5% of the housekeeping Na+/K+ ATPase on the cell surface, thereby providing direct evidence for a GSIS-dependent surface exposure of the ZnT8 self-antigen. Moreover, the variation in tagged-ZnT8 expression and surface labeling enabled sorting of heterogeneous beta cells to subpopulations that exhibited marked differences in GSIS with parallel changes in endogenous ZnT8 expression. The abundant surface display of endogenous ZnT8 and its coupling to GSIS demonstrated the potential of ZnT8 as a surface biomarker for tracking and isolating functional beta cells in mixed cell populations.

Lipid-tuned Zinc Transport Activity of Human ZnT8 Protein Correlates with Risk for Type-2 Diabetes.

Zinc is a critical element for insulin storage in the secretory granules of pancreatic beta cells. The islet-specific zinc transporter ZnT8 mediates granular sequestration of zinc ions. A genetic variant of human ZnT8 arising from a single nonsynonymous nucleotide change contributes to increased susceptibility to type-2 diabetes (T2D), but it remains unclear how the high risk variant (Arg-325), which is also a higher frequency (>50%) allele, is correlated with zinc transport activity. Here, we compared the activity of Arg-325 with that of a low risk ZnT8 variant (Trp-325). The Arg-325 variant was found to be more active than the Trp-325 form following induced expression in HEK293 cells. We further examined the functional consequences of changing lipid conditions to mimic the impact of lipid remodeling on ZnT8 activity during insulin granule biogenesis. Purified ZnT8 variants in proteoliposomes exhibited more than 4-fold functional tunability by the anionic phospholipids, lysophosphatidylcholine and cholesterol. Over a broad range of permissive lipid compositions, the Arg-325 variant consistently exhibited accelerated zinc transport kinetics versus the Trp-form. In agreement with the human genetic finding that rare loss-of-function mutations in ZnT8 are associated with reduced T2D risk, our results suggested that the common high risk Arg-325 variant is hyperactive, and thus may be targeted for inhibition to reduce T2D risk in the general populations.

The PP-motif in luminal loop 2 of ZnT transporters plays a pivotal role in TNAP activation.

Secretory and membrane-bound zinc-requiring enzymes are thought to be activated by binding zinc in the early secretory pathway. One such enzyme, tissue-non-specific alkaline phosphatase (TNAP), is activated through a two-step mechanism, via protein stabilization and subsequent enzyme activation through metalation, by ZnT5-ZnT6 heterodimers or ZnT7 homodimers. However, little is known about the molecular basis underlying the activation process. In the present study, we found that the di-proline motif (PP-motif) in luminal loop 2 of ZnT5 and ZnT7 is important for TNAP activation. TNAP activity was significantly reduced in cells lacking ZnT5-ZnT6 heterodimers and ZnT7 homodimers [triple knockout (TKO) cells]. The decreased TNAP activity was restored by expressing hZnT5 with hZnT6 or hZnT7, but significantly less so (almost 90% less) by expressing mutants thereof in which the PP-motif was mutated to alanine (PP-AA). In TKO cells, overexpressed hTNAP was not completely activated, and it was converted less efficiently into the holo form by expressing a PP-AA mutant of hZnT5 with hZnT6, whose defects were not restored by zinc supplementation. The zinc transport activity of hZnT7 was not significantly impaired by the PP-AA mutation, indicating that the PP-motif is involved in the TNAP maturation process, although it does not control zinc transport activity. The PP-motif is highly conserved in ZnT5 and ZnT7 orthologues, and its importance for TNAP activation is conserved in the Caenorhabditis elegans hZnT5 orthologue CDF5. These results provide novel molecular insights into the TNAP activation process in the early secretory pathway.

Visualizing the kinetic power stroke that drives proton-coupled zinc(II) transport.

The proton gradient is a principal energy source for respiration-dependent active transport, but the structural mechanisms of proton-coupled transport processes are poorly understood. YiiP is a proton-coupled zinc transporter found in the cytoplasmic membrane of Escherichia coli. Its transport site receives protons from water molecules that gain access to its hydrophobic environment and transduces the energy of an inward proton gradient to drive Zn(II) efflux. This membrane protein is a well-characterized member of the family of cation diffusion facilitators that occurs at all phylogenetic levels. Here we show, using X-ray-mediated hydroxyl radical labelling of YiiP and mass spectrometry, that Zn(II) binding triggers a highly localized, all-or-nothing change of water accessibility to the transport site and an adjacent hydrophobic gate. Millisecond time-resolved dynamics reveal a concerted and reciprocal pattern of accessibility changes along a transmembrane helix, suggesting a rigid-body helical re-orientation linked to Zn(II) binding that triggers the closing of the hydrophobic gate. The gated water access to the transport site enables a stationary proton gradient to facilitate the conversion of zinc-binding energy to the kinetic power stroke of a vectorial zinc transport. The kinetic details provide energetic insights into a proton-coupled active-transport reaction.

Intact functional fourteen-subunit respiratory membrane-bound [NiFe]-hydrogenase complex of the hyperthermophilic archaeon Pyrococcus furiosus.

The archaeon Pyrococcus furiosus grows optimally at 100 °C by converting carbohydrates to acetate, CO2, and H2, obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is encoded by a 14-gene operon with both hydrogenase and Na(+)/H(+) antiporter modules. Herein a His-tagged MBH was expressed in P. furiosus and the detergent-solubilized complex purified under anaerobic conditions by affinity chromatography. Purified MBH contains all 14 subunits by electrophoretic analysis (13 subunits were also identified by mass spectrometry) and had a measured iron:nickel ratio of 15:1, resembling the predicted value of 13:1. The as-purified enzyme exhibited a rhombic EPR signal characteristic of the ready nickel-boron state. The purified and membrane-bound forms of MBH both preferentially evolved H2 with the physiological donor (reduced ferredoxin) as well as with standard dyes. The O2 sensitivities of the two forms were similar (half-lives of ∼ 15 h in air), but the purified enzyme was more thermolabile (half-lives at 90 °C of 1 and 25 h, respectively). Structural analysis of purified MBH by small angle x-ray scattering indicated a Z-shaped structure with a mass of 310 kDa, resembling the predicted value (298 kDa). The angle x-ray scattering analyses reinforce and extend the conserved sequence relationships of group 4 enzymes and complex I (NADH quinone oxidoreductase). This is the first report on the properties of a solubilized form of an intact respiratory MBH complex that is proposed to evolve H2 and pump Na(+) ions.

Metalloproteomics: challenges and prospective for clinical research applications.

Metals are essential cofactors, utilized in many critical cellular processes. For example, zinc is important in insulin biosynthesis and may play a role in Alzheimer's disease, but much of how the zinc-mediated process remains unknown. Knowing which metal is in which protein at a given point in time would lead to new insights into how metals work in biological systems. New tools are being developed to investigate the biochemistry and cell biology of metals, with potential for biomedical applications. In this report, we consider the promise and limitations of metalloproteins detection techniques. We provide a brief overview of the techniques available and a discussion of the technical challenges to biomedical applications, with particular focus on what must be overcome for the potential of these approaches to be achieved.

Histidine pairing at the metal transport site of mammalian ZnT transporters controls Zn2+ over Cd2+ selectivity.

Zinc and cadmium are similar metal ions, but though Zn(2+) is an essential nutrient, Cd(2+) is a toxic and common pollutant linked to multiple disorders. Faster body turnover and ubiquitous distribution of Zn(2+) vs. Cd(2+) suggest that a mammalian metal transporter distinguishes between these metal ions. We show that the mammalian metal transporters, ZnTs, mediate cytosolic and vesicular Zn(2+) transport, but reject Cd(2+), thus constituting the first mammalian metal transporter with a refined selectivity against Cd(2+). Remarkably, the bacterial ZnT ortholog, YiiP, does not discriminate between Zn(2+) and Cd(2+). A phylogenetic comparison between the tetrahedral metal transport motif of YiiP and ZnTs identifies a histidine at the mammalian site that is critical for metal selectivity. Residue swapping at this position abolished metal selectivity of ZnTs, and fully reconstituted selective Zn(2+) transport of YiiP. Finally, we show that metal selectivity evolves through a reduction in binding but not the translocation of Cd(2+) by the transporter. Thus, our results identify a unique class of mammalian transporters and the structural motif required to discriminate between Zn(2+) and Cd(2+), and show that metal selectivity is tuned by a coordination-based mechanism that raises the thermodynamic barrier to Cd(2+) binding.

Selective electrodiffusion of zinc ions in a Zrt-, Irt-like protein, ZIPB.

All living cells need zinc ions to support cell growth. Zrt-, Irt-like proteins (ZIPs) represent a major route for entry of zinc ions into cells, but how ZIPs promote zinc uptake has been unclear. Here we report the molecular characterization of ZIPB from Bordetella bronchiseptica, the first ZIP homolog to be purified and functionally reconstituted into proteoliposomes. Zinc flux through ZIPB was found to be nonsaturable and electrogenic, yielding membrane potentials as predicted by the Nernst equation. Conversely, membrane potentials drove zinc fluxes with a linear voltage-flux relationship. Direct measurements of metal uptake by inductively coupled plasma mass spectroscopy demonstrated that ZIPB is selective for two group 12 transition metal ions, Zn(2+) and Cd(2+), whereas rejecting transition metal ions in groups 7 through 11. Our results provide the molecular basis for cellular zinc acquisition by a zinc-selective channel that exploits in vivo zinc concentration gradients to move zinc ions into the cytoplasm.

Structural basis for autoregulation of the zinc transporter YiiP.

Zinc transporters have crucial roles in cellular zinc homeostatic control. The 2.9-A resolution structure of the zinc transporter YiiP from Escherichia coli reveals a richly charged dimer interface stabilized by zinc binding. Site-directed fluorescence resonance energy transfer (FRET) measurements and mutation-activity analysis suggest that zinc binding triggers hinge movements of two electrically repulsive cytoplasmic domains pivoting around four salt bridges situated at the juncture of the cytoplasmic and transmembrane domains. These highly conserved salt bridges interlock transmembrane helices at the dimer interface, where they are well positioned to transmit zinc-induced interdomain movements to reorient transmembrane helices, thereby modulating coordination geometry of the active site for zinc transport. The cytoplasmic domain of YiiP is a structural mimic of metal-trafficking proteins and the metal-binding domains of metal-transporting P-type ATPases. The use of this common structural module to regulate metal coordination chemistry may enable a tunable transport activity in response to cytoplasmic metal fluctuations.

Structure of the zinc transporter YiiP.

YiiP is a membrane transporter that catalyzes Zn2+/H+ exchange across the inner membrane of Escherichia coli. Mammalian homologs of YiiP play critical roles in zinc homeostasis and cell signaling. Here, we report the x-ray structure of YiiP in complex with zinc at 3.8 angstrom resolution. YiiP is a homodimer held together in a parallel orientation through four Zn2+ ions at the interface of the cytoplasmic domains, whereas the two transmembrane domains swing out to yield a Y-shaped structure. In each protomer, the cytoplasmic domain adopts a metallochaperone-like protein fold; the transmembrane domain features a bundle of six transmembrane helices and a tetrahedral Zn2+ binding site located in a cavity that is open to both the membrane outer leaflet and the periplasm.