High-affinity Zinc Binding Site Research Articles

Binding of two zinc ions promotes liquid-liquid phase separation of Tau

Tau is a naturally disordered microtubule associated protein which forms intraneuronal aggregates in several neurodegenerative diseases including Alzheimer's disease (AD). It was reported that zinc interaction with tau protein can trigger its aggregation. Recently we identified three zinc binding sites located in the N-terminal part, repeat region and the C-terminal part of tau. Here we characterized zinc binding to each of the three sites using isothermal titration calorimetry (ITC) and determined the impact of each site on aggregation using dynamic light scattering (DLS) assays. First, we confirmed the presence of three zinc binding sites on tau and determined the thermodynamic parameters of binding of zinc to these sites. We found a high-affinity zinc binding site located in the repeat region of tau and two N- and C-terminus binding sites with a lower binding constant for zinc. Second, we showed that tau aggregation necessitates zinc binding to the high affinity site in the R2R3 region, while LLPS necessitates zinc binding to any two binding sites. With regard to the role of zinc ions in the aggregation of proteins in neurodegenerative diseases, these findings bring new insights to the understanding of the aggregation mechanism of tau protein induced by zinc.

Inhibition of an organophosphate-detoxifying bacterial phosphotriesterase by albumin and plasma thiol components

The phosphotriesterase of the bacterium Brevundimonas diminuta (BdPTE) is a naturally occurring enzyme that catalyzes the hydrolysis of organophosphate (OP) nerve agents as well as pesticides and offers a potential treatment of corresponding intoxications. While BdPTE mutants with improved catalytic efficiencies against several OPs have been described, unexpectedly, less efficient breakdown of an OP was observed upon application in an animal model compared with in vitro measurements. Here, we describe detailed inhibition studies with the high-activity BdPTE mutant 10-2C3(C59M/C227A) by human plasma components, indicating that this enzyme is inhibited by serum albumin. The inhibitory activity is mediated by depletion of crucial zinc ions from the BdPTE active site, either via the known high-affinity zinc binding site of albumin or via chemical complex formation with its free thiol side chain at position Cys34. Albumin pre-charged with zinc ions or carrying a chemically blocked Cys34 side chain showed significantly reduced inhibitory activity; in fact, the combination of both measures completely abolished BdPTE inhibition. Consequently, the available zinc ion concentration in blood plays an important role for BdPTE activity in vivo and should be taken into account for therapeutic development and application of a catalytic OP scavenger.

Engineered high-affinity zinc binding site reveals gating configurations of a human proton channel.

The voltage-gated proton channel (HV1) is a voltage sensor that also conducts protons. The singular ability of protons to penetrate proteins complicates distinguishing closed and open channels. When we replaced valine with histidine at position 116 in the external vestibule of hHV1, current was potently inhibited by externally applied Zn2+ in a construct lacking the two His that bind Zn2+ in WT channels. High-affinity binding with profound effects at 10 nM Zn2+ at pHo 7 suggests additional groups contribute. We hypothesized that Asp185, which faces position 116 in our closed-state model, contributes to Zn2+ chelation. Confirming this prediction, V116H/D185N abolished Zn2+ binding. Studied in a C-terminal truncated monomeric construct, V116H channels activated rapidly. Anomalously, Zn2+ slowed activation, producing a time constant independent of both voltage and Zn2+ concentration. We hypothesized that slow turn-on of H+ current in the presence of Zn2+ reflects the rate of Zn2+ unbinding from the channel, analogous to drug-receptor dissociation reactions. This behavior in turn suggests that the affinity for Zn2+ is greater in the closed state of hHV1. Supporting this hypothesis, pulse pairs revealed a rapid component of activation whose amplitude decreased after longer intervals at negative voltages as closed channels bound Zn2+. The lower affinity of Zn2+ in open channels is consistent with the idea that structural rearrangements within the transmembrane region bring Arg205 near position 116, electrostatically expelling Zn2+. This phenomenon provides direct evidence that Asp185 opposes position 116 in closed channels and that Arg205 moves between them when the channel opens.

Crystal structures of AztD provide mechanistic insights into direct zinc transfer between proteins

Zinc acquisition from limited environments is critical for bacterial survival and pathogenesis. AztD has been identified as a periplasmic or cell surface zinc-binding protein in numerous bacterial species. In Paracoccus denitrificans, AztD can transfer zinc directly to AztC, the solute binding protein for a zinc-specific ATP-binding cassette transporter system, suggesting a role in zinc acquisition and homeostasis. Here, we present the first cry\tstal structures of AztD from P. denitrificans and tbe human pathogen Citrobacter koseri, revealing a beta-propeller fold and two high-affinity zinc-binding sites that are highly conserved among AztD homologs. These structures combined with transfer assays using WT and mutant proteins provide rare insight into the mechanism of direct zinc transfer from one protein to another. Given the importance of zinc import to bacterial pathogenesis, these insights may prove valuable to the development of zinc transfer inhibitors as antibiotics.

Copper and zinc ions govern the trans-directed dimerization of APP family members in multiple ways.

The amyloid precursor protein (APP) and its homologs amyloid precursor-like protein1 (APLP1) and APLP2 have central physiological functions in transcellular adhesion that depend on copper and zinc mediated trans-directed dimerization of the extracellular domains E1 and E2. Copper binds to three distinct sites in APP, one in the copper binding (CuBD) and growthfactor-like (GFLD) domains each within E1, and one in the E2 domain. For APLP1 and APLP2, metal binding has so far only been shown for the E2 domain. Zinc binding has been reported for all APP family members to a unique site in the E2 domain and an additional site essential for APLP1 E2 domain trans-dimerization. Using isothermal titration calorimetry, co-immunoprecipitation, and invitro bead aggregation assays, we show that copper promotes cis- as well as trans-directed dimerization of APLP1 and APLP2, similar as reported previously for APP. Furthermore, we report a APP-specific zinc binding site with nanomolar affinity located in the E1 domain, whereas no binding of zinc to the individual subdomains GFLD or CuBD was detected. Zinc binding did not affect the cis- but trans-dimerization of APP and APLP1. Furthermore, zinc binding inhibited copper-induced trans-directed dimerization of APP. Together, we identified a high-affinity APP-specific zinc binding site in the E1 domain and revealed contrasting cis- and trans-directed dimerization properties of APP, APLP1, and APLP2 in dependence on zinc and copper ions. Consequently, changes in metal ion homeostasis, as reported in the context of synaptic activity and neurodegenerative diseases, appear as key modulators of homo- and heterotypic trans-cellular APP/APLPs complexes.

Mechanisms of zinc binding to the solute-binding protein AztC and transfer from the metallochaperone AztD

Bacteria can acquire the essential metal zinc from extremely zinc-limited environments by using ATP-binding cassette (ABC) transporters. These transporters are critical virulence factors, relying on specific and high-affinity binding of zinc by a periplasmic solute-binding protein (SBP). As such, the mechanisms of zinc binding and release among bacterial SBPs are of considerable interest as antibacterial drug targets. Zinc SBPs are characterized by a flexible loop near the high-affinity zinc-binding site. The function of this structure is not always clear, and its flexibility has thus far prevented structural characterization by X-ray crystallography. Here, we present intact structures for the zinc-specific SBP AztC from the bacterium Paracoccus denitrificans in the zinc-bound and apo-states. A comparison of these structures revealed that zinc loss prompts significant structural rearrangements, mediated by the formation of a sodium-binding site in the apo-structure. We further show that the AztC flexible loop has no impact on zinc-binding affinity, stoichiometry, or protein structure, yet is essential for zinc transfer from the metallochaperone AztD. We also found that 3 His residues in the loop appear to temporarily coordinate zinc and then convey it to the high-affinity binding site. Thus, mutation of any of these residues to Ala abrogated zinc transfer from AztD. Our structural and mechanistic findings conclusively identify a role for the AztC flexible loop in zinc acquisition from the metallochaperone AztD, yielding critical insights into metal binding by AztC from both solution and AztD. These proteins are highly conserved in human pathogens, making this work potentially useful for the development of novel antibiotics.

Molecular Basis for Subtype Specificity and High-Affinity Zinc Inhibition in the GluN1-GluN2A NMDA Receptor Amino-Terminal Domain

Zinc is vastly present in the mammalian brain and controls functions of various cell surface receptors to regulate neurotransmission. A distinctive characteristic of N-methyl-D-aspartate (NMDA) receptors containing a GluN2A subunit is that their ion channel activity is allosterically inhibited by a nano-molar concentration of zinc that binds to an extracellular domain called an amino-terminal domain (ATD). Despite physiological importance, the molecular mechanism underlying the high-affinity zinc inhibition has been incomplete because of the lack of a GluN2A ATD structure. Here we show the first crystal structures of the heterodimeric GluN1-GluN2A ATD, which provide the complete map of the high-affinity zinc-binding site and reveal distinctive features from the ATD of the GluN1-GluN2B subtype. Perturbation of hydrogen bond networks at the hinge of the GluN2A bi-lobe structure affects both zinc inhibition and open probability, supporting the general model in which the bi-lobe motion in ATD regulates the channel activity in NMDA receptors.

Modulation of extrasynaptic NMDA receptors by synaptic and tonic zinc

Many excitatory synapses contain high levels of mobile zinc within glutamatergic vesicles. Although synaptic zinc and glutamate are coreleased, it is controversial whether zinc diffuses away from the release site or whether it remains bound to presynaptic membranes or proteins after its release. To study zinc transmission and quantify zinc levels, we required a high-affinity rapid zinc chelator as well as an extracellular ratiometric fluorescent zinc sensor. We demonstrate that tricine, considered a preferred chelator for studying the role of synaptic zinc, is unable to efficiently prevent zinc from binding low-nanomolar zinc-binding sites, such as the high-affinity zinc-binding site found in NMDA receptors (NMDARs). Here, we used ZX1, which has a 1 nM zinc dissociation constant and second-order rate constant for binding zinc that is 200-fold higher than those for tricine and CaEDTA. We find that synaptic zinc is phasically released during action potentials. In response to short trains of presynaptic stimulation, synaptic zinc diffuses beyond the synaptic cleft where it inhibits extrasynaptic NMDARs. During higher rates of presynaptic stimulation, released glutamate activates additional extrasynaptic NMDARs that are not reached by synaptically released zinc, but which are inhibited by ambient, tonic levels of nonsynaptic zinc. By performing a ratiometric evaluation of extracellular zinc levels in the dorsal cochlear nucleus, we determined the tonic zinc levels to be low nanomolar. These results demonstrate a physiological role for endogenous synaptic as well as tonic zinc in inhibiting extrasynaptic NMDARs and thereby fine tuning neuronal excitability and signaling.

Mutational Analysis of the High-Affinity Zinc Binding Site Validates a Refined Human Dopamine Transporter Homology Model

The high-resolution crystal structure of the leucine transporter (LeuT) is frequently used as a template for homology models of the dopamine transporter (DAT). Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i) when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii) LeuT and DAT share a rather low overall sequence identity (22%) and (iii) the extracellular loop 2 (EL2) of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter‚s movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

A comparative study on the possible zinc binding sites of the human ZnT3 zinc transporter protein

The brain specific zinc transporter protein ZnT3 can be related to the amyloid neuropathology of Alzheimer's disease. In order to analyze the metal binding ability of human ZnT3 protein, here we report a potentiometric and solution structural (UV-Vis, CD, EPR, NMR) study of nickel(II), copper(II) and zinc(II) complexes of three peptides mimicking the possible metal binding sequences of this protein. The peptide L¹ (Ac-RHQAGPPHSHR-NH2) is a minimalist, the cyclic peptide L² (cyclo(Ac-CKLHQAGPPHSHGSRGAEYAPLEEGPEEKC-NH2) is a more complete model of the intracellular His-rich loop, which is widely accepted as a putative metal binding site. The peptide L³ (Ac-PFHHCHRD-NH2) is the model of the conserved cytoplasmic N-terminal -HHCH- sequence. In the physiological pH-range, the ZnL¹, ZnH3L² and ZnL³ complexes are the major species in the corresponding binary systems, with {3N(im)}, {3N(im),2/3O(amide)} and {3N(im),S(-)} coordination environments, respectively. The species ZnL³ has 3-4 orders of magnitude higher stability than the other two complexes, indicating the presence of a high-affinity zinc-binding site at the N-terminal tail of the human ZnT3 transporter. Moreover, L³ shows preferred zinc binding as compared to nickel (log β(ZnL³) - log β(NiL³) = 2.3), probably due to the higher preference of zinc(II) for tetrahedral geometry. These facts suggest that zinc binding to the N-terminal -HHCH- sequence of human ZnT3 may be involved in the biological activity of this zinc transporter protein in zinc sensing, binding or translocation processes.

The Zinc Dyshomeostasis Hypothesis of Alzheimer's Disease

Alzheimer's disease (AD) is the most common form of dementia in the elderly. Hallmark AD neuropathology includes extracellular amyloid plaques composed largely of the amyloid-β protein (Aβ), intracellular neurofibrillary tangles (NFTs) composed of hyper-phosphorylated microtubule-associated protein tau (MAP-tau), and microtubule destabilization. Early-onset autosomal dominant AD genes are associated with excessive Aβ accumulation, however cognitive impairment best correlates with NFTs and disrupted microtubules. The mechanisms linking Aβ and NFT pathologies in AD are unknown. Here, we propose that sequestration of zinc by Aβ-amyloid deposits (Aβ oligomers and plaques) not only drives Aβ aggregation, but also disrupts zinc homeostasis in zinc-enriched brain regions important for memory and vulnerable to AD pathology, resulting in intra-neuronal zinc levels, which are either too low, or excessively high. To evaluate this hypothesis, we 1) used molecular modeling of zinc binding to the microtubule component protein tubulin, identifying specific, high-affinity zinc binding sites that influence side-to-side tubulin interaction, the sensitive link in microtubule polymerization and stability. We also 2) performed kinetic modeling showing zinc distribution in extra-neuronal Aβ deposits can reduce intra-neuronal zinc binding to microtubules, destabilizing microtubules. Finally, we 3) used metallomic imaging mass spectrometry (MIMS) to show anatomically-localized and age-dependent zinc dyshomeostasis in specific brain regions of Tg2576 transgenic, mice, a model for AD. We found excess zinc in brain regions associated with memory processing and NFT pathology. Overall, we present a theoretical framework and support for a new theory of AD linking extra-neuronal Aβ amyloid to intra-neuronal NFTs and cognitive dysfunction. The connection, we propose, is based on β-amyloid-induced alterations in zinc ion concentration inside neurons affecting stability of polymerized microtubules, their binding to MAP-tau, and molecular dynamics involved in cognition. Further, our theory supports novel AD therapeutic strategies targeting intra-neuronal zinc homeostasis and microtubule dynamics to prevent neurodegeneration and cognitive decline.

Role of Zinc in the Pathogenesis of Attention-Deficit Hyperactivity Disorder

The dopamine transporter is regulated by zinc (Zn2+), which directly interacts with the transporter protein as a potent non-competitive blocker of substrate translocation (dopamine transport inward and outward). The fact that dysfunction of the dopamine transporter is involved in the pathogenesis of attention-deficit hyperactivity disorder (ADHD) is interesting in the context of studies that suggest the involvement of zinc deficiency in patients with ADHD. In this article, we present a hypothesis exploring the causative mechanism of zinc deficiency in ADHD and why zinc might be beneficial as a supplementary medication and/or adjunct to psychostimulants (methylphenidate, amfetamine) in zinc-deficient ADHD patients. The hypothesis is based on published in vitro observations that the human dopamine transporter contains a high-affinity zinc binding site (His-193, His-375, Glu-396) on its extracellular face that modulates transporter function, and in vivo studies suggesting that response to stimulants is reduced in zinc-deficient ADHD patients. It seems likely that zinc supplementation in zinc-deficient ADHD patients improves the binding status of insufficiently occupied zinc binding sites on the dopamine transporter. We propose to test our hypothesis by recruiting zinc-deficient ADHD patients who will undergo positron emission tomography with the 11C-raclopride displacement method to investigate whether zinc increases extracellular dopamine levels.

Albumin as a zinc carrier: properties of its high-affinity zinc-binding site

Although details of the molecular mechanisms for the uptake of the essential nutrient zinc into the bloodstream and its subsequent delivery to zinc-requiring organs and cells are poorly understood, it is clear that in vertebrates the majority of plasma zinc (9-14 microM; approx. 75-85%) is bound to serum albumin, constituting part of the so-called exchangeable pool. The binding of metal ions to serum albumins has been the subject of decades of studies, employing a multitude of techniques, but only recently has the identity and putative structure of the major zinc site on albumin been reported. Intriguingly, this site is located at the interface between two domains, and involves two residues from each of domains I and II. Comparisons of X-ray crystal structures of free and fatty-acid bound human serum albumin suggest that zinc binding to this site and fatty acid binding to one of the five major sites may be interdependent. Interactive binding of zinc and long-chain fatty acids to albumin may therefore have physiological implications.

Interaction of Pseudomonas putida CZ1 with clays and ability of the composite to immobilize copper and zinc from solution

The present study was conducted to determine the abilities of the living and nonliving Pseudomonas putida CZ1 cells, clays (goethite, kaolinite, smectite and manganite) and their composites to accumulate copper and zinc from a liquid medium, and elucidate the role of microbes on the mobility of heavy metals. Various mixtures of bacteria and clays were exposed to solutions of 0.025 mM or 0.5 mM Cu(II) and Zn(II) in 0.01 M KNO 3 to differentiate between so-called “high-affinity” sites and “low-affinity” sites. Clays associated in an edge-on orientation to the cells was observed by electron microscope (EM) examination of these metal-treated bacteria–clay aggregates. Adsorption experiments and desorption with 1.0 M CH 3COOK solution indicated that clays contain more high-affinity copper binding sites and less high-affinity zinc binding sites than that of bacteria, however, bacteria are involved in more low-affinity heavy-metal-binding sites. Carboxyl group activity is more important at weak-binding sites than at strong-binding sites. TEM-EDS analysis confirmed that most of Zn removed from solution was associated with P. putida CZ1 in the composites. These results suggest that bacteria play an important role in regulating the mobility of heavy metals in the soil environment.

Possible Regulatory Role for the Histidine-Rich Loop in the Zinc Transport Protein, ZnuA,

A number of bacterial metal transporters belong to the ABC transporter family. To better understand the structural determinants of metal selectivity of one such transporter, we previously determined the structure of the periplasmic domain of a zinc transporter, ZnuA, from Synechocystis 6803 and found that ZnuA binds zinc via three histidines. Unique to these ABC zinc transporters, ZnuA has a highly charged and mobile loop that protrudes from the protein in the vicinity of the metal binding site that we had suggested might facilitate zinc acquisition. To further examine the function of this loop, the structure and zinc binding properties of two ZnuA variants were determined. When the loop is entirely deleted, zinc still binds to the three histidines. However, unlike what was suggested from the structure of a similar solute binding protein, TroA, release of zinc occurs concomitantly with large conformational changes in two of the three chelating histidines. These structural results combined with isothermal titration calorimetry data demonstrate that there are at least two classes of zinc binding sites: the high-affinity site in the cleft between the two domains and at least one additional site on the flexible loop. This loop has approximately 100-fold weaker affinity for zinc than the high-affinity zinc binding site, and its deletion does not affect the high-affinity site. From these results, we suggest that this region might be a sensor for high periplasmic levels of zinc.

Zinc-buffering capacity of a eukaryotic cell at physiological pZn

In spite of the paramount importance of zinc in biology, dynamic aspects of cellular zinc metabolism remain poorly defined at the molecular level. Investigations with human colon cancer (HT-29) cells establish a total cellular zinc concentration of 264 microM. Remarkably, about 10% of the potential high-affinity zinc-binding sites are not occupied by zinc, resulting in a surplus of 28 muM ligands (average Kd(c) = 83 pM) that ascertain cellular zinc-buffering capacity and maintain the "free" zinc concentration in proliferating cells at picomolar levels (784 pM, pZn = 9.1). This zinc-buffering capacity allows zinc to fluctuate only with relatively small amplitudes (DeltapZn = 0.3; below 1 nM) without significantly perturbing physiological pZn. Thus, the "free" zinc concentrations in resting and differentiated HT-29 cells are 614 pM and 1.25 nM, respectively. The calculation of these "free" zinc concentrations is based on measurements at different concentrations of the fluorogenic zinc-chelating agent and extrapolation to a zero concentration of the agent. It depends on the state of the cell, its buffering capacity, and the zinc dissociation constant of the chelating agent. Zinc induction of thionein (apometallothionein) ensures a surplus of unbound ligands, increases zinc-buffering capacity and the availability of zinc (DeltapZn = 0.8), but preserves the zinc-buffering capacity of the unoccupied high-affinity zinc-binding sites, perhaps for crucial physiological functions. Jointly, metallothionein and thionein function as the major zinc buffer under conditions of increased cellular zinc.

Thioredoxin 2, an Oxidative Stress-induced Protein, Contains a High Affinity Zinc Binding Site

Two thioredoxins have been described in Escherichia coli, TrxA and Trx2. Both thioredoxins are capable of reducing disulfide bonds using a conserved pair of cysteine residues present in a WCGPC motif. A number of unique structural and regulatory features distinguish the Trx2 subfamily from the much larger TrxA family. The Trx2 subfamily has an additional N-terminal domain of +/- 30 residues, which contains two additional conserved CXXC motifs. Moreover, the gene coding for Trx2 is under control of the oxidative stress transcription factor OxyR in E. coli. This suggests that Trx2 may play a role in the cellular defense against oxidative stress. We show here that Trx2 contains zinc in a 1:1 stoichiometry, making it the first identified zinc-binding thioredoxin. The zinc atom is coordinated by the four cysteines of the two N-terminal CXXC motifs. The zinc center of Trx2 binds zinc with a very high affinity (Ka of >1018 m-1). We show that in vitro oxidation of the zinc binding cysteines by H2O2 releases the zinc and induces a conformational change. The zinc-free protein conserves its reductase activity. Altogether, our results suggest that the zinc center might play the role of a redox switch, changing a yet to be identified activity.

Functional role, structure, and evolution of the melanocortin-4 receptor.

The melanocortin (MC)-4 receptor participates in regulating body weight homeostasis. We demonstrated early that acute blockage of the MC-4 receptor increases food intake and relieves anorexic conditions in rats. Our recent studies show that 4-week chronic blockage of the MC-4 receptor leads to robust increases in food intake and development of obesity, whereas stimulation of the receptor leads to anorexia. Interestingly, the food conversion ratio was clearly increased by MC-4 receptor blockage, whereas it was decreased in agonist-treated rats in a transient manner. Chronic infusion of an agonist caused a transient increase in oxygen consumption. Our studies also show that the MC-4 receptor plays a role in luteinizing hormone and prolactin surges in female rats. The MC-4 receptor has a role in mediating the effects of leptin on these surges. The phylogenetic relation of the MC-4 receptor to other GPCRs in the human genome was determined. The three-dimensional structure of the protein was studied by construction of a high-affinity zinc binding site between the helices, using two histidine residues facing each other. We also cloned the MC-4 receptor from evolutionary important species and showed by chromosomal mapping a conserved synteny between humans and zebrafish. The MC-4 receptor has been remarkably conserved in structure and pharmacology for more than 400 million years, implying that the receptor participated in vital physiological functions early in vertebrate evolution.

Effect of zinc and temperature on the conformation of the gamma subunit of retinal phosphodiesterase: a natively unfolded protein.

The cyclic GMP phosphodiesterase gamma-subunit (PDEgamma) was shown to belong to the family of natively unfolded proteins. Increasing temperature transforms the protein into a more ordered (but still relatively disordered) conformation. The C-terminal part of PDEgamma has a high-affinity zinc-binding site (Kd approximately 1 microM), with His75 and His79 being directly involved into the coordination of Zn2+. Zinc-loaded protein remains effectively unfolded. Possible implications of these findings to the functioning of PDEgamma are discussed.

Zinc binding in intestinal brush-border membrane isolated from pig

Zinc binding to brush-border membrane vesicles isolated from pig jejunum was investigated by a rapid filtration method, for long incubation periods (up to 180 min). Zn 2+ influx revealed a large accumulation of the metal, reaching an apparent intravesicular volume of 160 μl/mg protein at equilibrium, a volume 45-times that of an osmotically reactive sugar, sorbitol (3.6 μl/mg protein). Changes in medium osmolarity had no effect on zinc accumulation. These results suggested a large degree of zinc binding to vesicular components (membrane or core). 65Zn efflux measurements led to the conclusion that two vesicular pools of zinc existed: a small external pool, accessible to different chelators (EGTA) or competetive cations, and a large intravesicular pool. Accumulated 65Zn was quickly removed from its internal sites only after the membrane had been permeabilized by the cation ionophore A23187 in association with an exchange molecule or a chelator. Scatchard plot analyses revealed, on one hand a first class of high-affinity extravesicular zinc binding sites ( K a = 8.6 · 10 3M −1, n = 0.455nmol Zn 2+/mg protein) and a second class of extravesicular sites having a very low affinity ( K a = 22M −1, n = 25.35nmol Zn 2+/mg protein) and, on the other hand one type of intravesicular sites ( K a = 3.3 · 10 4M −1, n = 550 nmol Zn 2+/mg protein). The intravesicular sites have a high affinity for zinc and are specific, since only nonlabelled zinc (or cadmium) but not calcium present in the bathing medium is exchanged with the internally accumulated labelled cation.