A novel clan of zinc metallopeptidases with possible intramembrane cleavage properties

Computer-based database searching and protein multiple sequence alignment has identified a novel clan of zinc metallopeptidases, which, by phylogenetic analysis, has been shown to contain six subfamilies. The family is characterized by four common transmembrane segments and three conserved sequence motifs. The combination of topology analysis and motif identification has detected three potential Zn2+ coordinating residues. Only two of the sequences of this novel zinc metallopeptidase clan possess any functional annotation, one of which is able to cleave its substrate within a cytosol/transmembrane segment junction. A number of observations suggest that the remaining members of this novel clan may also cleave their substrates within transmembrane segments.     

A tetrahedral coordination of Zinc during transmembrane transport by P-type Zn(2+)-ATPases

Zn(2+) is an essential transition metal required in trace amounts by all living organisms. However, metal excess is cytotoxic and leads to cell damage. Cells rely on transmembrane transporters, with the assistance of other proteins, to establish and maintain Zn(2+) homeostasis. Metal coordination during transport is key to specific transport and unidirectional translocation without the backward release of free metal. The coordination details of Zn(2+) at the transmembrane metal binding site responsible for transport have now been established. Escherichia coli ZntA is a well-characterized Zn(2+)-ATPase responsible for intracellular Zn(2+) efflux. A truncated form of the protein lacking regulatory metal sites and retaining the transport site was constructed. Metrical parameters of the metal-ligand coordination geometry for the zinc bound isolated form were characterized using x-ray absorption spectroscopy (XAS). Our data support a nearest neighbor ligand environment of (O/N)(2)S(2) that is compatible with the proposed invariant metal coordinating residues present in the transmembrane region. This ligand identification and the calculated bond lengths support a tetrahedral coordination geometry for Zn(2+) bound to the TM-MBS of P-type ATPase transporters.     

The LZT proteins; the LIV-1 subfamily of zinc transporters

Zinc is an essential ion for cells with a vital role to play in controlling the cellular processes of the cell, such as growth, development and differentiation. Specialist proteins called zinc transporters control the level of intracellular zinc in cells. In mammals, the ZIP family of zinc transporters has a pivotal role in maintaining the correct level of intracellular zinc by their ability to transport zinc into cells from outside, although they may also transport metal ions other than zinc. There are now recognised to be four subfamilies of the ZIP transporters, including the recently discovered LIV-1 subfamily which has similarity to the oestrogen-regulated gene LIV-1, previously implicated in metastatic breast cancer. We call this new subfamily LZT, for LIV-1 subfamily of ZIP zinc Transporters. Here we document current knowledge of this previously uncharacterised group of proteins, which includes the KE4 proteins. LZT proteins are similar to ZIP transporters in secondary structure and ability to transport metal ions across the plasma membrane or intracellular membranes. However, LZT proteins have a unique motif (HEXPHEXGD) with conserved proline and glutamic acid residues, unprecedented in other zinc transporters. The localisation of LZT proteins to lamellipodiae mirrors cellular location of the membrane-type matrix metalloproteases. These differences to other zinc transporters may be consistent with an alternative role for LZT proteins in cells, particularly in diseases such as cancer.     

Metal selectivity determinants in a family of transition metal transporters

Metal tolerance proteins (MTPs) are plant members of the cation diffusion facilitator (CDF) transporter family involved in cellular metal homeostasis. Members of the CDF family are ubiquitously found in all living entities and show principal selectivity for Zn(2+), Mn(2+), and Fe(2+). Little is known regarding metal selectivity determinants of CDFs. We identified a novel cereal member of CDFs in barley, termed HvMTP1, that localizes to the vacuolar membrane. Unlike its close relative AtMTP1, which is highly selective for Zn(2+), HvMTP1 exhibits selectivity for both Zn(2+) and Co(2+) as assessed by its ability to suppress yeast mutant phenotypes for both metals. Expression of HvMTP1/AtMTP1 chimeras in yeast revealed a five-residue sequence within the AtMTP1 N-segment of the His-rich intracytoplasmic loop that confines specificity to Zn(2+). Furthermore, mutants of AtMTP1 generated through random mutagenesis revealed residues embedded within transmembrane domain 3 that additionally specify the high degree of Zn(2+) selectivity. We propose that the His-rich loop, which might play a role as a zinc chaperone, determines the identity of the metal ions that are transported. The residues within transmembrane domain 3 can also influence metal selectivity, possibly through conformational changes induced at the cation transport site located within the membrane or at the cytoplasmic C-terminal domain.     

Characteristics of zinc transport by two bacterial cation diffusion facilitators from Ralstonia metallidurans CH34 and Escherichia coli

CzcD from Ralstonia metallidurans and ZitB from Escherichia coli are prototypes of bacterial members of the cation diffusion facilitator (CDF) protein family. Expression of the czcD gene in an E. coli mutant strain devoid of zitB and the gene for the zinc-transporting P-type ATPase zntA rendered this strain more zinc resistant and caused decreased accumulation of zinc. CzcD, purified as an amino-terminal streptavidin-tagged protein, bound Zn2+, Co2+, Cu2+, and Ni2+ but not Mg2+, Mn2+, or Cd2+, as shown by metal affinity chromatography. Histidine residues were involved in the binding of 2 to 3 mol of Zn2+ per mol of CzcD. ZitB transported 65Zn2+ in the presence of NADH into everted membrane vesicles with an apparent Km of 1.4 microM and a Vmax of 0.57 nmol of Zn2+ min(-1) mg of protein(-1). Conserved amino acyl residues that might be involved in binding and transport of zinc were mutated in CzcD and/or ZitB, and the influence on Zn2+ resistance was studied. Charged or polar amino acyl residues that were located within or adjacent to membrane-spanning regions of the proteins were essential for the full function of the proteins. Probably, these amino acyl residues constituted a pathway required for export of the heavy metal cations or for import of counter-flowing protons.     

A role for ZnT-1 in regulating cellular cation influx

The ZnTs are a growing family of proteins involved in lowering or sequestration of cellular zinc. Using fluorescent measurements of zinc transport we have addressed the mechanism of action of the most ubiquitously expressed member of this family, ZnT-1. This protein has been shown to lower levels of intracellular zinc though the mechanism has remained elusive. The rate of zinc efflux in HEK293 cells expressing ZnT-1 was not accelerated in comparison to control cells, suggesting that ZnT-1 may be involved in regulating influx rather than efflux of zinc. Co-expression of the L-type calcium channel, a major route for zinc influx, and ZnT-1 resulted in a 3-fold reduction in the rate of zinc influx in HEK293 and PC-12 cells, indicating that ZnT-1 modulates zinc permeation through this channel. Immunoblot analysis indicates that ZnT-1 expression does not modulate LTCC expression. Our findings therefore indicate that ZnT-1 modulates the permeation of cations through LTCC, thereby, regulating cation homeostasis through this pathway. Furthermore, ZnT-1 may play a role in cellular ion homeostasis and thereby confer protection against pathophysiological events linked to cellular Ca(2+) or Zn(2+) permeation and cell death.     

A sodium zinc exchange mechanism is mediating extrusion of zinc in mammalian cells

Zinc influx, driven by a steep inward electrochemical gradient, plays a fundamental role in zinc signaling and in pathophysiologies linked to intracellular accumulation of toxic zinc. Yet, the cellular transport mechanisms that actively generate or maintain the transmembrane gradients are not well understood. We monitored Na+-dependent Zn2+ transport in HEK293 cells and cortical neurons, using fluorescent imaging. Treatment of the HEK293 cells with CaPO4 precipitates induced Na+-dependent Zn2+ extrusion, against a 500-fold transmembrane zinc gradient, or zinc influx upon reversal of Na+ gradient, thus indicating that Na+/Zn2+ exchange is catalyzing active Zn2+ transport. Depletion of intracellular ATP did not inhibit the Na+-dependent Zn2+ extrusion, consistent with a mechanism involving a secondary active transporter. Inhibitors of the Na+/Ca2+ exchanger failed to inhibit Na+-dependent Zn2+ efflux. In addition, zinc transport was unchanged in HEK293 cells heterologously expressing functional cardiac or neuronal Na+/Ca2+ exchangers, thus indicating that the Na+/Zn2+ exchange activity is not mediated by the Na+/Ca2+ exchanger. Sodium-dependent zinc exchange, facilitating the removal of intracellular zinc, was also monitored in neurons. To our knowledge, the Na+/Zn2+ exchanger described here is the first example of a mammalian transport mechanism capable of Na+-dependent active extrusion of zinc. Such mechanism is likely to play an important role, not only in generating the transmembrane zinc gradients, but also in protecting cells from the potentially toxic effects of permeation of this ion.     

Annexin VI regulation of cardiac function

Annexins are a family of membrane binding proteins that are characterized by a hypervariable amino terminus followed by a series of highly conserved Ca2+-phospholipid binding domains. Annexins function by binding to anionic phospholipid surfaces in a Ca2+-dependent manner. They self-associate to form trimers which further assemble into sheets that cover the membrane surface and alter properties such as fluidity and permeability. This submembranous skeleton alters integral protein functions such as ion transport properties and shields the surface from phospholipid binding proteins such as phospholipases and protein kinase C. Transgenic mouse hearts overexpressing wild type annexin VI (AnxVI673), a dominant-negative truncated annexin VI (residues 1-129, Anx129) and an annexin VI-null mouse (AnxVI-/-) have implicated the protein as a regulator of intracellular Ca2+ homeostasis which affects cardiac function.     

Isolation of an intestinal metallothionein induced by parenteral zinc

An intestinal zinc-binding protein, induced by parenteral zinc administration, has been isolated and characterized. Based upon its elution behavior in two chromatographic systems, Zn²⁺/protein ratio of 5.0–5.6 gram atoms/mole, a Zn²⁺/SH ratio of about 3.0, paucity of both aromatic amino acids and absorbance at 280 nm, abundance of cysteic acid residues (28–31%), and low molecular weight (6,000–7,000 daltons), the protein meets the criteria for classification as a metallothionein and is more properly named zinc-thionein. Orally administered ⁶⁵Zn was found to bind to intestinal zinc-thionein and thus this intracellular protein may function as a component of the mechanism responsible for mammalian zinc homeostasis at the level of intestinal absorption.     

Mapping the zinc ligands of S100A2 by site-directed mutagenesis

S100 family proteins are characterized by short individual N and C termini and a conserved central part, harboring two Ca(2+)-binding EF-hands, one of them highly conserved among EF-hand family proteins and the other characteristic for S100 proteins. In addition to Ca(2+), several members of the S100 protein family, including S100A2, bind Zn(2+). Two regions in the amino acid sequences of S100 proteins, namely the helices of the N-terminal EF-hand motif and the very C-terminal loop are believed to be involved in Zn(2+)-binding due to the presence of histidine and/or cysteine residues. Human S100A2 contains four cysteine residues, each of them located at positions that may be important for Zn(2+) binding. We have now constructed and purified 10 cysteine-deficient mutants of human S100A2 by site-directed mutagenesis and investigated the contribution of the individual cysteine residues to Zn(2+) binding. Here we show that Cys(1(3)) (the number in parentheses indicating the position in the sequence of S100A2) is the crucial determinant for Zn(2+) binding in association with conformational changes as determined by internal tyrosine fluorescence. Solid phase Zn(2+) binding assays also revealed that the C-terminal residues Cys(3(87)) and Cys(4(94)) mediated a second type of Zn(2+) binding, not associated with detectable conformational changes in the molecule. Cys(2(22)), by contrast, which is located within the first EF hand motif affected neither Ca(2+) nor Zn(2+) binding, and a Cys "null" mutant was entirely incapable of ligating Zn(2+). These results provide new information about the mechanism and the site(s) of zinc binding in S100A2.     

ZntB is a novel Zn2+ transporter in Salmonella enterica serovar Typhimurium

A Zn2+ transport system encoded by the zntB locus of Salmonella enterica serovar Typhimurium has been identified. The protein encoded by this locus is homologous to the CorA family of Mg2+ transport proteins and is widely distributed among the eubacteria. Mutations at zntB confer an increased sensitivity to the cytotoxic effects of Zn2+ and Cd2+, a phenotype that suggests that the encoded protein mediates the efflux of both cations. A direct analysis of transport activity identified a capacity for Zn2+ efflux. These data identify ZntB as a zinc efflux pathway in the enteric bacteria and assign a new function to the CorA family of cation transporters.     

Structure-function analysis of the zinc-binding region of the Clpx molecular chaperone

The ClpX heat shock protein of Escherichia coli is a member of the universally conserved Hsp100 family of proteins, and possesses a putative zinc finger motif of the C(4) type. The ClpX is an ATPase which functions both as a substrate specificity component of the ClpXP protease and as a molecular chaperone. Using an improved purification procedure we show that the ClpX protein is a metalloprotein complexed with Zn(II) cations. Contrary to other Hsp100 family members, ClpXZn(II) exists in an oligomeric form even in the absence of ATP. We show that the single ATP-binding site of ClpX is required for a variety of tasks, namely, the stabilization of the ClpXZn(II) oligomeric structure, binding to ClpP, and the ClpXP-dependent proteolysis of the lambdaO replication protein. Release of Zn(II) from ClpX protein affects the ability of ClpX to bind ATP. ClpX, free of Zn(II), cannot oligomerize, bind to ClpP, or participate in ClpXP-dependent proteolysis. We also show that ClpXDeltaCys, a mutant protein whose four cysteine residues at the putative zinc finger motif have been replaced by serine, behaves in similar fashion as wild type ClpX protein whose Zn(II) has been released either by denaturation and renaturation, or chemically by p-hydroxymercuriphenylsulfonic acid.     

Biochemical properties of vacuolar zinc transport systems of Saccharomyces cerevisiae

The yeast vacuole plays an important role in zinc homeostasis by storing zinc for later use under deficient conditions, sequestering excess zinc for its detoxification, and buffering rapid changes in intracellular zinc levels. The mechanisms involved in vacuolar zinc sequestration are only poorly characterized. Here we describe the properties of zinc transport systems in yeast vacuolar membrane vesicles. The major zinc transport activities in these vesicles were ATP-dependent, requiring a H+ gradient generated by the V-ATPase for function. One system we identified was dependent on the ZRC1 gene, which encodes a member of the cation diffusion facilitator family of metal transporters. These data are consistent with the proposed role of Zrc1 as a vacuolar zinc transporter. Zrc1-independent activity was also observed that was not dependent on the closely related vacuolar Cot1 protein. Both Zrc1-dependent and independent activities showed a high specificity for Zn(2+) over other physiologically relevant substrates such as Ca2+, Fe2+, and Mn2+. Moreover, these systems had high affinities for zinc with apparent K(m) values in the 100-200 nm range. These results provide biochemical insight into the important role of Zrc1 and related proteins in eukaryotic zinc homeostasis.     

Conserved aspartic acid 714 in transmembrane segment 8 of the ZntA subgroup of P1B-type ATPases is a metal-binding residue

ZntA from Escherichia coli is a member of the P1B-type ATPase family that confers resistance specifically to Pb2+, Zn2+, and Cd2 salts by active efflux across the cytoplasmic membrane. P1B-type ATPases are important for homeostasis of metal ions such as Cu+, Ag+, Pb2+, Zn2+, Cd2+ Cu2+, and Co2+, with different subgroups showing specificity for different metal ions. Sequence alignments of P1B-type ATPases show that ZntA and close homologues have a strictly conserved Asp714 in the eighth transmembrane domain that is not conserved in other subgroups of P1B-type ATPases. However, in the sarcoplasmic reticulum Ca2+-ATPase, a structurally characterized P-type ATPase, the residue corresponding to Asp714 is a metal-binding residue. Four site-specific mutants at Asp714, D714E, D714H, D714A, and D714P, were characterized. A comparison of their metal-binding affinity with that of wtZntA revealed that Asp714 is a ligand for the metal ion in the transmembrane site. Thus, Asp714 is one of the residues that determine metal ion specificity in ZntA homologues. All four substitutions at Asp714 in ZntA resulted in complete loss of in vivo resistance activity and complete or large reductions in ATPase activity, though D714E and D714H retained the ability to bind metal ions with high affinity at the transmembrane site. Thus, the ability to bind metal ions with high affinity did not correlate with high activity. The metal-binding affinity of the N-terminal site remained unchanged in all four mutants. The affinities of the two metal-binding sites in wtZntA determined in this study are similar to values reported previously for the individual sites in isolated ZntA fragments.     

Identification of a novel family of targets of PYK2 related to Drosophila retinal degeneration B (rdgB) protein

The protein tyrosine kinase PYK2 has been implicated in signaling pathways activated by G-protein-coupled receptors, intracellular calcium, and stress signals. Here we describe the molecular cloning and characterization of a novel family of PYK2-binding proteins designated Nirs (PYK2 N-terminal domain-interacting receptors). The three Nir proteins (Nir1, Nir2, and Nir3) bind to the amino-terminal domain of PYK2 via a conserved sequence motif located in the carboxy terminus. The primary structures of Nirs reveal six putative transmembrane domains, a region homologous to phosphatidylinositol (PI) transfer protein, and an acidic domain. The Nir proteins are the human homologues of the Drosophila retinal degeneration B protein (rdgB), a protein implicated in the visual transduction pathway in flies. We demonstrate that Nirs are calcium-binding proteins that exhibit PI transfer activity in vivo. Activation of PYK2 by agents that elevate intracellular calcium or by phorbol ester induce tyrosine phosphorylation of Nirs. Moreover, PYK2 and Nirs exhibit similar expression patterns in several regions of the brain and retina. In addition, PYK2-Nir complexes are detected in lysates prepared from cultured cells or from brain tissues. Finally, the Nir1-encoding gene is located at human chromosome 17p13.1, in proximity to a locus responsible for several human retinal diseases. We propose that the Nir and rdgB proteins represent a new family of evolutionarily conserved PYK2-binding proteins that play a role in the control of calcium and phosphoinositide metabolism downstream of G-protein-coupled receptors.     

An antiport mechanism for a member of the cation diffusion facilitator family: divalent cations efflux in exchange for K+ and H+

Members of the cation diffusion facilitator (CDF) family of membrane transport proteins are found in eukaryotes and prokaryotes. The family encompasses transporters of zinc ions, with cobalt, cadmium and lead ions being additional substrates for some prokaryotic examples. No transport mechanism has previously been established for any CDF protein. It is shown here that the CzcD protein of Bacillus subtilis, a CDF protein, uses an antiporter mechanism, catalysing active efflux of Zn2+ in exchange for K+ and H+. The exchange is probably electroneutral, energized by the transmembrane pH gradient and oppositely oriented gradients of the other cation substrates. The data suggest that Co2+ and Cd2+ are additional cytoplasmic substrates for CzcD. A second product of the same operon that encodes czcD has sequence similarity to oxidoreductases and is here designated CzcO. CzcO modestly enhances the activity of CzcD but is not predicted to be an integral membrane protein and has no antiport activity of its own.     

Solution structure of the Mycobacterium tuberculosis EsxG·EsxH complex: functional implications and comparisons with other M. tuberculosis Esx family complexes

Mycobacterium tuberculosis encodes five type VII secretion systems that are responsible for exporting a number of proteins, including members of the Esx family, which have been linked to tuberculosis pathogenesis and survival within host cells. The gene cluster encoding ESX-3 is regulated by the availability of iron and zinc, and secreted protein products such as the EsxG·EsxH complex have been associated with metal ion acquisition. EsxG and EsxH have previously been shown to form a stable 1:1 heterodimeric complex, and here we report the solution structure of the complex, which features a core four-helix bundle decorated at both ends by long, highly flexible, N- and C-terminal arms that contain a number of highly conserved residues. Despite clear similarities in the overall backbone fold to the EsxA·EsxB complex, the structure reveals some striking differences in surface features, including a potential protein interaction site on the surface of the EsxG·EsxH complex. EsxG·EsxH was also found to contain a specific Zn(2+) binding site formed from a cluster of histidine residues on EsxH, which are conserved across obligate mycobacterial pathogens including M. tuberculosis and Mycobacterium leprae. This site may reflect an essential role in zinc ion acquisition or point to Zn(2+)-dependent regulation of its interaction with functional partner proteins. Overall, the surface features of both the EsxG·EsxH and the EsxA·EsxB complexes suggest functions mediated via interactions with one or more target protein partners.     

Identification of Helical Packing Motifs Common to Bacteriorhodopsin and G Protein-Coupled Receptors

A sequence analysis and comparison of transmembrane helices in bacteriorhodopsin (BR) and G protein-coupled receptors (GPCRs) is presented to identify potential regions of homology across protein families. The results show a common pattern of residues is conserved within the interhelical contact regions of BR that fit a knob-into-hole structural motif previously postulated for globular proteins and photosynthetic reaction centers. Based on an alignment of conserved prolines in transmembrane helices, it is inferred that analogous helix packing arrangements are possible in the rhodopsin-like GPCRs. Molecular models of GPCR helices V and VI indicate these interactions occur between aromatic and hydrophobic residues flanking the highly conserved prolines in these sequences. A similar packing arrangement is shown to occur in the X-ray structure of the melittin which also displays a unique pairing of proline-linked helices. The contact pattern identified is further applied to predict the packing of pairs of proline-containing helices in the pheromone-like and cAMP GPCRs. A potential role in stabilizing structure formation is also suggested for the contacts. The results and conclusions are supported by recent biophysical studies of zinc binding to kappa-opioid receptor mutants.