Allosteric negative regulation of smt O/P binding of the zinc sensor,SmtB, by metal ions: a coupled equilibrium analysis

The Synechococcus PCC 7942 smt operon is responsible for cellular resistance to excess zinc and consists of two divergently transcribed genes, smtB and smtA. SmtB is the Zn(II)-sensing metal-regulated repressor of the system and binds to a 12-2-12 imperfect inverted repeat in the smtA O/P region. Using fluorescence anisotropy to monitor SmtB-smt O/P multiple equilibria, we show that four SmtB homodimers bind to a 40 bp oligonucleotide containing a single 12-2-12 inverted repeat. The binding affinities of the first two dimers are very tight (K(int) = 2.9 x 10(9) M(-1)) with the affinities of the third and fourth dimers lower by approximately 10- and approximately 30-fold, respectively. A single monomer equivalent of Zn(II), Cd(II), or Co(II) promotes disassembly of the oligomeric complex to a mixture of (P(2)).D and (P(2))(2).D SmtB dimer-DNA complexes with the intrinsic affinity of all SmtB homodimers for DNA greatly reduced by approximately 500-2000-fold. Substitution or derivatization of cysteines which comprise the alpha3N metal binding site (Cys14 and Cys61) [VanZile, M. L., et al. (2002) Biochemistry 41, 9765-9775] has no effect on allosteric negative regulation by Zn(II); in contrast, H106Q SmtB, harboring a single zinc-liganding substitution in the alpha5 metal binding site, is refractory to zinc-induced disassembly of SmtB-DNA complexes. The alpha5 metal binding sites are therefore regulatory for Zn(II) sensing in vitro and in vivo, while the high-affinity alpha3N sites play some other role. This finding for SmtB is the opposite of that previously determined for Staphylococcus aureus pI258 CadC, a Pb(II)/Cd(II)/Bi(III) sensor [Busenlehner, L. S., et al. (2002) J. Mol. Biol. 319, 685-701], thus providing insight into the origin of functional metal ion selectivity in this family of metal sensor proteins.     

SmtB-DNA and protein-protein interactions in the formation of the cyanobacterial metallothionein repression complex: Zn2+ does not dissociate the protein-DNA complex in vitro.

The synechococcal metallothionein locus smt consists of two divergent genes: smtA coding for the metallothionein SmtA, and smtB coding for the trans-acting regulator SmtB. The latter binds at two inverted repeats, designated S1/S2 and S3/S4, in the overlapping promoter/operator sites between the two genes. We have determined the binding stoichiometries to the entire operator/promoter DNA and to the separate S1/S2 and S3/S4 half-operator oligonucleotides using sedimentation equilibrium and sedimentation velocity measurements. The full promoter/operator DNA binds two SmtB dimers. The hydrodynamic behavior of this complex supports a compact nucleoprotein structure. Each separate S1/S2 and S3/S4 operator sequence also binds two dimers. An equal molar mixture of separate S1/S2 and S3/S4 operator sequences, in excess SmtB, forms a S1/S2-SmtB:SmtB-S3/S4 bridge complex. Combining these results with previously published binding interference data, which showed consecutive S1/S2 and S3/S4 SmtB occupancy on the operator/promoter DNA, we have developed a model for the establishment of the repression complex that appears to involve significant DNA compaction, presumably DNA bending, stabilized by SmtB-SmtB bridge interactions. DNase I footprinting titrations also showed consecutive S1/S2 and S3/S4 SmtB occupancy. The footprints expand considerably in the presence of Zn2+. Hence, SmtB remains bound to the operator sites when Zn2+ ions are present. This result is further supported by gel retardation assay. Failure of the metal ions to dissociate SmtB from the DNA points to a hitherto unknown function of SmtB in the regulation of the smt locus.     

Cyanobacterial metallothionein gene expressed in Escherichia coli. Metal-binding properties of the expressed protein.

The recently isolated Synechococcus gene smtA encodes the only characterised prokaryotic protein designated to be a metallothionein (MT). To examine the metal-binding properties of its product the smtA gene was expressed in Escherichia coli as a carboxyterminal extension of glutathione-S-transferase. The pH of half dissociation of Zn, Cd and Cu ions from the expressed protein was determined to be 4.10, 3.50, 2.35, respectively, indicating a high affinity for these ions (in particular for Zn in comparison to mammalian MT). E. coli expressing this gene showed enhanced (ca. 3-fold) accumulation of Zn.     

A novel cyanobacterial SmtB/ArsR family repressor regulates the expression of a CPx-ATPase and a metallothionein in response to both Cu(I)/Ag(I) and Zn(II)/Cd(II)

A novel SmtB/ArsR family metalloregulator, denoted BxmR, has been identified and characterized from the cyanobacterium Oscillatoria brevis. Genetic and biochemical evidence reveals that BxmR represses the expression of both bxa1, encoding a CPx-ATPase metal transporter, as well as a divergently transcribed operon encoding bxmR and bmtA, a heavy metal sequestering metallothionein. Derepression of the expression of all three genes is mediated by both monovalent (Ag(I) and Cu(I)) and divalent (Zn(II) and Cd(II)) heavy metal ions, a novel property among SmtB/ArsR metal sensors. Electrophoretic gel mobility shift experiments reveal that apoBxmR forms multiple resolvable complexes with oligonucleotides containing a single 12-2-12 inverted repeat derived from one of the two operator/promoter regions with similar apparent affinities. Preincubation with either monovalent or divalent metal ions induces disassembly of both the BxmR-bxa1 and BxmR-bxmR/bmtA operator/promoter complexes. Interestingly, the temporal regulation of expression of bxa1 and bmtA mRNAs is different in O. brevis with bxa1 induced first upon heavy metal treatment, followed by bmtA/bxmR. A dynamic interplay among Bxa1, BmtA, and BxmR is proposed that maintains metal homeostasis in O. brevis by balancing the relative rates of metal storage and efflux of multiple heavy metal ions.     

Zn2+ selectively stabilizes FdU-substituted DNA through a unique major groove binding motif

We report, based on semi-empirical calculations, that Zn(2+) binds duplex DNA containing consecutive FdU-dA base pairs in the major groove with distorted trigonal bipyramidal geometry. In this previously uncharacterized binding motif, O4 and F5 on consecutive FdU are axial ligands while three water molecules complete the coordination sphere. NMR spectroscopy confirmed Zn(2+) complexation occurred with maintenance of base pairing while a slight hypsochromic shift in circular dichroism (CD) spectra indicated moderate structural distortion relative to B-form DNA. Zn(2+) complexation inhibited ethidium bromide (EtBr) intercalation and stabilized FdU-substituted duplex DNA (ΔT(m) > 15 °C). Mg(2+) neither inhibited EtBr complexation nor had as strong of a stabilizing effect. DNA sequences that did not contain consecutive FdU were not stabilized by Zn(2+). A lipofectamine preparation of the Zn(2+)-DNA complex displayed enhanced cytotoxicity toward prostate cancer cells relative to the individual components prepared as lipofectamine complexes indicating the potential utility of Zn(2+)-DNA complexes for cancer treatment.     

Studies on the kinetic stabilities of the Gd(3+) complexes formed with the N-mono(methylamide), N'-mono(methylamide) and N,N"-bis(methylamide) derivatives of diethylenetriamine-N,N,N',N",N"-pentaacetic acid

High kinetic stability is an important requirement for the Gd(3+) complexes used as contrast enhancement agents in magnetic resonance imaging. The kinetic stabilities of the Gd(3+) complexes formed with DTPA-N-mono(methylamide) (L(3)), DTPA-N'-mono(methylamide) (L(2)) and DTPA-bis(methylamide) (L(1)) are characterized by the rates of the exchange reactions with Eu(3+) and the endogenous Cu(2+) and Zn(2+). The exchange reactions occur via the proton-assisted dissociation of the complexes and direct attack of the exchanging metal ions on the complex. On the basis of the line-shape analysis of the 1H NMR spectra of the LaL(2), obtained in the pH range 2.5-3.5, we assume that for the proton-assisted dissociation of the complexes the formation of an intermediate containing a free iminodiacetate group must be followed with the rupture of the metal-central nitrogen bond. At about pH > or = 5, the reactions between GdL(2) or GdL(3) and Cu(2+) or Zn(2+) proceed predominantly by direct reaction of the reactants, through the formation of dinuclear intermediates. The contribution of the proton-assisted dissociation is highly important for GdL(1), but its reaction with Zn(2+) is significantly slower than the reactions of GdL(2) and GdL(3). The overall rates of dissociation of GdL(1), GdL(2), GdL(3) and Gd(DTPA)(2-) through H(+) (pH 7.4), Cu(2+) (1 x 10(-6) M) and Zn(2+) (1 x 10(-5) M)-assisted reactions are surprisingly very similar. Replacement of one or two carboxylates with amide groups results in significantly decreased stability constants, but has practically no effect on the kinetic stability of the Gd(3+) complexes, indicating the lower reactivity of the amide groups with Cu(2+) and Zn(2+).     

Interactions of bismuth complexes with metallothionein(II).

Bismuth complexes are widely used as anti-ulcer drugs and can significantly reduce the side effects of platinum anti-cancer drugs. Bismuth is known to induce the synthesis of metallothionein (MT) in the kidney, but there are few chemical studies on the interactions of bismuth complexes with metallothionein. Here we show that Bi(3+) binds strongly to metallothionein with a stoichiometry bismuth:MT = 7:1 (Bi(7)MT) and can readily displace Zn(2+) and Cd(2+). Bismuth is still bound to the protein even in strongly acidic solutions (pH 1). Reactions of bismuth citrate with MT are faster than those of [Bi(EDTA)](-), and both exhibit biphasic kinetics. (1)H NMR data show that Zn(2+) is displaced faster than Cd(2+), and that both Zn(2+) and Cd(2+) in the beta-domain (three metal cluster) of MT are displaced by Bi(3+) much faster than from the alpha-domain (four metal cluster). The extended x-ray absorption fine structure spectrum of Bi(7)MT is very similar to that for the glutathione and N-acetyl-L-cysteine complexes [Bi(GS)(3)] and [Bi(NAC)(3)] with an inner coordination sphere of three sulfur atoms and average Bi-S distances of 2.55 A. Some sites appear to contain additional short Bi-O bonds of 2.2 A and longer Bi-S bonds of 3.1 A. The Bi(3+) sites in Bi(7)MT are therefore highly distorted in comparison with those of Zn(2+) and Cd(2+).     

Ca(2+) and Zn(2+) binding properties of peptide substrates of vertebrate collagenase, MMP-1.

To understand the role of Ca(2+) in vertebrate in the structure and action of collagenase, we have examined peptides that interact with recombinant human fibroblast collagenase for their affinities towards Ca(2+) and Zn(2+) in a non-polar solvent. Two of the peptides, GPQGIAGQ and GNVGLAGA, had sequences in collagen which are, respectively, cleaved and not cleaved by collagenase. A third peptide, PSYFLNAG, had a collagenase-cleaved sequence in ovostatin, a globular protein substrate. Peptides TVGCEECTV and CLPREPGL were derived from TIMP-1; the former competitively inhibits collagenase while the latter does not. The relative rates of hydrolysis of the peptides by collagenase had the order GPQGIAGQ>PSYFLNAG>GNVGLAGA. Circular dichroism spectral data in trifluoroethanol showed that while the TIMP control peptide, CLPREPGL, bound only Zn(2+), the other four peptides bound both Ca(2+) and Zn(2+) with definite stoichiometries. Ca(2+) could displace Zn(2+) in the substrate peptides while Zn(2+) displaced Ca(2+) in the TIMP peptide. GPQGIAGQ, PSYFLNAG and TVGCEECTV formed peptide:Ca(2+):Zn(2+) ternary complexes. Our results suggest that both collagen and globular protein substrates of collagenase may bind Ca(2+) and Zn(2+) in the enzyme's active site. This, in turn, may account for the known importance of the non-catalytic Ca(2+) and Zn(2+) in collagenase activity.     

Crystal structures of fructose 1,6-bisphosphatase: mechanism of catalysis and allosteric inhibition revealed in product complexes

Crystal structures of metal-product complexes of fructose 1, 6-bisphosphatase (FBPase) reveal competition between AMP and divalent cations. In the presence of AMP, the Zn(2+)-product and Mg(2+)-product complexes have a divalent cation present only at one of three metal binding sites (site 1). The enzyme is in the T-state conformation with a disordered loop of residues 52-72 (loop 52-72). In the absence of AMP, the enzyme crystallizes in the R-state conformation, with loop 52-72 associated with the active site. In structures without AMP, three metal-binding sites are occupied by Zn(2+) and two of three metal sites (sites 1 and 2) by Mg(2+). Evidently, the association of AMP with FBPase disorders loop 52-72, the consequence of which is the release of cations from two of three metal binding sites. In the Mg(2+) complexes (but not the Zn(2+) complexes), the 1-OH group of fructose 6-phosphate (F6P) coordinates to the metal at site 1 and is oriented for a nucleophilic attack on the bound phosphate molecule. A mechanism is presented for the forward reaction, in which Asp74 and Glu98 together generate a hydroxide anion coordinated to the Mg(2+) at site 2, which then displaces F6P. Development of negative charge on the 1-oxygen of F6P is stabilized by its coordination to the Mg(2+) at site 1.     

Determination of the structure of Escherichia coli glyoxalase I suggests a structural basis for differential metal activation

The metalloenzyme glyoxalase I (GlxI) converts the nonenzymatically produced hemimercaptal of cytotoxic methylglyoxal and glutathione to nontoxic S-D-lactoylglutathione. Human GlxI, for which the structure is known, is active in the presence of Zn(2+). Unexpectedly, the Escherichia coli enzyme is inactive in the presence of Zn(2+) and is maximally active with Ni(2+). To understand this difference in metal activation and also to obtain a representative of the bacterial enzymes, the structure of E. coli Ni(2+)-GlxI has been determined. Structures have also been determined for the apo enzyme as well as complexes with Co(2+), Cd(2+), and Zn(2+). It is found that each of the protein-metal complexes that is catalytically active has octahedral geometry. This includes the complexes of the E. coli enzyme with Ni(2+), Co(2+), and Cd(2+), as well as the structures reported for the human Zn(2+) enzyme. Conversely, the complex of the E. coli enzyme with Zn(2+) has trigonal bipyramidal coordination and is inactive. This mode of coordination includes four protein ligands plus a single water molecule. In contrast, the coordination in the active forms of the enzyme includes two water molecules bound to the metal ion, suggesting that this may be a key feature of the catalytic mechanism. A comparison of the human and E. coli enzymes suggests that there are differences between the active sites that might be exploited for therapeutic use.     

Stability and transmetallation of the magnetic resonance contrast agent MnDPDP measured by EPR

MnDPDP [manganese(II) N, N'-dipyridoxylethylenediamine- N, N'-diacetate-5,5'-bis(phosphate)] is the active component of Teslascan, a contrast medium for magnetic resonance imaging of the liver. It has previously been shown that MnDPDP is rapidly dephosphorylated to the monophosphate MnDPMP and the non-phosphorylated MnPLED, and that all these substances are rapidly transmetallated to the corresponding Zn complexes. In the present study we used EPR at 9 and 230 GHz to show that no free Mn(2+) ions can be detected in the product or in a mixture of MnDPDP and human serum. Competition experiments between MnDPDP and Zn(2+), Ca(2+), and Mg(2+) ions revealed approximately 15% transmetallation with Zn(2+) in a buffer system containing metal ion concentrations similar to that in serum, whereas approximately 10% transmetallation was obtained with Ca(2+) and only negligible transmetallation was obtained with Mg(2+) under these conditions. Binding experiments with Mn(2+) added to human albumin and human serum indicate that albumin accounts for most of the protein-bound Mn(2+) in serum.     

A Brownian dynamics study of the interaction of Phormidium laminosum plastocyanin with Phormidium laminosum cytochrome f

The interaction of Phormidium laminosum plastocyanin (PC) with P. laminosum cytochrome f (cyt f) was studied using Brownian dynamics (BD) simulations. Few complexes and a low rate of electron transfer were observed for wild-type PC. Increasing the positive electrostatic field on PC by the addition of a Zn(2+) ion in the neighborhood of D44 and D45 on PC (as found in crystal structure of plastocyanin) increased the number of complexes formed and the calculated rates of electron transfer as did PC mutations D44A, D45A, E54A, and E57A. Mutations of charged residues on Phormidium PC and Phormidium cyt f were used to map binding sites on both proteins. In both the presence and absence of the Zn(2+) ion, the following residues on PC interact with cyt f: D44, D45, K6, D79, R93, and K100 that lie in a patch just below H92 and Y88 and D10, E17, and E70 located on the upper portion of the PC molecule. In the absence of the Zn(2+) ion, K6 and K35 on the top of the PC molecule also interact with cyt f. Cyt f residues involved in binding PC, in the absence of the Zn(2+) ion, include E165, D187, and D188 that are located on the small domain of cyt f. The orientation of PC in the complexes was quite random in accordance with NMR results. In the presence of the Zn(2+) ion, K53 and E54 in the lower patch of the PC molecule also interact with cyt f and PC interacts with E86, E95, and E123 on the large domain of cyt f. Also, the orientation of PC in the complexes was much more uniform than in the absence of the Zn(2+) ion. The difference may be due to both the larger electrostatic field and the greater asymmetry of the charge distribution on PC observed in the presence of the Zn(2+) ion. Hydrophobic interactions were also observed suggesting a model of cyt f-PC interactions in which electrostatic forces bring the two molecules together but hydrophobic interactions participate in stabilizing the final electron-transfer-active dock.