Comparison of the modes of action of a Vero toxin (a Shiga-like toxin) from Escherichia coli, of ricin, and of alpha-sarcin.

The modes of action of a Vero toxin (VT2 or Shiga-like toxin II) from Escherichia coli, of ricin, and of alpha-sarcin were compared. Elongation factor 1 (EF1) and GTP-dependent Phe-tRNA binding to ribosomes in the presence of poly(U) was inhibited by these three toxins, but EF1 and guanylyl (beta, gamma-methylene)-diphosphate-dependent Phe-tRNA binding was inhibited by alpha-sarcin only. EF1- and Phe-tRNA-dependent GTPase activity was inhibited by these toxins, but nonenzymatic binding of Phe-tRNA was not. The turnover rate of EF1 binding to ribosomes during Phe-tRNA binding was also decreased by these three toxins. The addition of EF1 recovered the inhibition of Phe-tRNA binding to ribosomes by VT2 and ricin but not by alpha-sarcin. The formation of and EF2- and GTP-dependent puromycin derivative of phenylalanine was inhibited slightly by the three toxins, indicating that translocation is not influenced significantly by them. EF2-dependent GTPase activity was stimulated by these toxins, and especially by VT2 and ricin. In contrast, the binding of EF2 to ribosomes was inhibited strongly by VT2 and ricin, and slightly by alpha-sarcin. The stimulation of EF2-dependent GTPase activity by the toxins may compensate for the decrease of EF2 binding to ribosomes which they caused during translocation. In total, these results indicate that VT2 and ricin inhibit protein synthesis through the disturbance of the turnover of EF1 binding to ribosomes during aminoacyl-tRNA binding to ribosomes, and that alpha-sarcin inhibits the synthesis through the inhibition of the binding of the complex of Phe-tRNA, EF1, and GTP to ribosomes.     

DNA binding to mica correlates with cationic radius: assay by atomic force microscopy.

In buffers containing selected transition metal salts, DNA binds to mica tightly enough to be directly imaged in the buffer in the atomic force microscope (AFM, also known as scanning force microscope). The binding of DNA to mica, as measured by AFM-imaging, is correlated with the radius of the transition metal cation. The transition metal cations that effectively bind DNA to mica are Ni(II), Co(II), and Zn(II), which have ionic radii from 0.69 to 0.74 A. In Mn(II), ionic radius 0.82 A, DNA binds weakly to mica. In Cd(II) and Hg(II), respective ionic radii of 0.97 and 1.1 A, DNA does not bind to mica well enough to be imaged with the AFM. These results may to relate to how large a cation can fit into the cavities above the recessed hydroxyl groups in the mica lattice, although hypotheses based on hydrated ionic radii cannot be ruled out. The dependence of DNA binding on the concentrations of the cations Ni(II), Co(II), or Zn(II) shows maximal DNA binding at approximately 1-mM cation. Mg(II) does not bind DNA tightly enough to mica for AFM imaging. Mg(II) is a Group 2 cation with an ionic radius similar to that of Ni(II). Ni(II), Co(II), and Zn(II) have anomalously high enthalpies of hydration that may relate to their ability to bind DNA to mica. This AFM assay for DNA binding to mica has potential applications for assaying the binding of other polymers to mica and other flat surfaces.     

Genetically engineered metal ion binding sites on the outside of a Channel's transmembrane beta-barrel.

We are exploring the ability of genetically engineered versions of the Staphylococcus aureus alpha-hemolysin (alphaHL) ion channel to serve as rationally designed sensor components for analytes including divalent cations. We show here that neither the hemolytic activity nor the single channel current of wild-type alphaHL was affected by [Zn(II)] </= 1 mM. Binding sites for the divalent cations were formed by altering the number and location of coordinating side chains, e.g., histidines and aspartic acids, between positions 126 and 134, inclusive. Several mutant alphaHLs exhibited Zn(II)-induced current noise that varied with Zn(II) concentration. At a fixed divalent cation concentration, the current fluctuation kinetics depended on the analyte type, e.g., Zn(II), Cu(II), Ni(II), and Co(II). We also show that the ability of Zn(II) to change the mutant channel current suggests that the pore's topology is beta-sheet and that position 130 is near the turn at the trans mouth. Both conclusions are consistent with the crystal structure of WT-alphaHL oligomerized in detergent. Our results, in the context of the channel's crystal structure, suggest that conductance blockades were caused by Zn(II) binding to the outside surface of the pore. Thus, analyte-induced current blockades alone might not establish whether an analyte binding site is inside a pore.     

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.     

Expression of mammalian Rab Escort protein-1 and -2 in yeast Saccharomyces cerevisiae

A simple method for preparation of alpha-sarcin and an antifungal protein (AFP) from mold (Aspergillus giganteus MDH 18894) has been developed. alpha-Sarcin and AFP were purified simultaneously by chitin affinity column chromatography and gel filtration. By this method, 4.5 mg of pure alpha-sarcin and 6.9 mg of pure AFP were obtained from 2 liters of culture medium. Compared with other purification methods such as ion-exchange column chromatography, this procedure was very simple and specific. The purified alpha-sarcin and AFP were homogeneous as characterized by SDS-polyacrylamide gel electrophoresis. Both alpha-sarcin and AFP exhibited the binding activity to generated chitin. Soluble glycochitin decreased the intensity of fluorescence of alpha-sarcin and made the lambda(em)m shift from 340 to 347 nm. Titration of alpha-sarcin with N-bromosuccinimide under native conditions revealed that two tryptophans (Trps) were all located in the core part of alpha-sarcin molecule. This indicated that Trps were not involved in the binding of alpha-sarcin to chitin. Glycochitin in the culture medium increased the expression of alpha-sarcin, while it had no effect on the expression of AFP. Unlike other ligands such as Cibacron blue for the affinity purification of alpha-sarcin and AFP, glycochitin increased the nuclease activity of alpha-sarcin.     

Study of the interaction between the antitumour protein alpha-sarcin and phospholipid vesicles.

alpha-Sarcin is a single polypeptide chain protein which exhibits antitumour activity by degrading the larger ribosomal RNA of tumour cells. We describe the interaction of a alpha-sarcin with lipid model systems. The protein specifically interacts with negatively-charged phospholipid vesicles, resulting in protein-lipid complexes which can be isolated by ultracentrifugation in a sucrose gradient. alpha-Sarcin causes aggregation of such vesicles. The extent of this interaction progressively decreases when the molar ratio of phosphatidylcholine increases in acidic vesicles. The kinetics of the vesicle aggregation induced by the protein have been measured. This process is dependent on the ratio of alpha-sarcin present in the protein-lipid system. A saturation plot is observed from phospholipid vesicles-protein titrations. The saturating protein/lipid molar ratio is 1:50. The effect produced by the antitumour protein on the lipid vesicles is dependent on neither the length nor the degree of unsaturation of the phospholipid acyl chain. However, the aggregation is dependent on temperature, being many times higher above the phase transition temperature of the corresponding phospholipid than below it. The effects of pH and ionic strength have also been considered. An increase in the ionic strength does not abolish the protein-lipid interaction. The effect of pH may be related to conformational changes of the protein. Binding experiments reveal a strong interaction between alpha-sarcin and acidic vesicles, with Kd = 0.06 microM. The peptide bonds of the protein are protected against trypsin hydrolysis upon binding to acidic vesicles. The interaction of the protein with phosphatidylglycerol vesicles does not modify the phase transition temperature of the lipid, although it decreases the amplitude of the change of fluorescence anisotropy associated to the co-operative melting of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labelled vesicles. The results are interpreted in terms of the existence of both electrostatic and hydrophobic components for the interaction between phospholipid vesicles and the antitumour protein.     

Arginine 121 is a crucial residue for the specific cytotoxic activity of the ribotoxin alpha-sarcin.

Alpha-sarcin, a cyclizing ribonuclease secreted by the mould Aspergillus giganteus, is one of the best characterized members of a family of fungal ribotoxins. This protein induces apoptosis in tumour cells due to its highly specific activity on ribosomes. Fungal ribotoxins display a three-dimensional protein fold similar to those of a larger group of microbial noncytotoxic RNases, represented by RNases T1 and U2. This similarity involves the three catalytic residues and also the Arg121 residue, whose counterpart in RNase T1, Arg77, is located in the vicinity of the substrate phosphate moiety although its potential functional role is not known. In this work, Arg121 of alpha-sarcin has been replaced by Gln or Lys. These two mutations do not modify the conformation of the protein but abolish the ribosome-inactivating activity of alpha-sarcin. In addition, the loss of the positive charge at that position produces dramatic changes on the interaction of alpha-sarcin with phospholipid membranes. It is concluded that Arg121 is a crucial residue for the characteristic cytotoxicity of alpha-sarcin and presumably of the other fungal ribotoxins.     

Metal selectivity of the Escherichia coli nickel metallochaperone, SlyD

SlyD is a Ni(II)-binding protein that contributes to nickel homeostasis in Escherichia coli. The C-terminal domain of SlyD contains a rich variety of metal-binding amino acids, suggesting broader metal binding capabilities, and previous work demonstrated that the protein can coordinate several types of first-row transition metals. However, the binding of SlyD to metals other than Ni(II) has not been previously characterized. To improve our understanding of the in vitro metal-binding activity of SlyD and how it correlates with the in vivo function of this protein, the interactions between SlyD and the series of biologically relevant transition metals [Mn(II), Fe(II), Co(II), Cu(I), and Zn(II)] were examined by using a combination of optical spectroscopy and mass spectrometry. Binding of SlyD to Mn(II) or Fe(II) ions was not detected, but the protein coordinates multiple ions of Co(II), Zn(II), and Cu(I) with appreciable affinity (K(D) values in or below the nanomolar range), highlighting the promiscuous nature of this protein. The order of affinities of SlyD for the metals examined is as follows: Mn(II) and Fe(II) < Co(II) < Ni(II) ~ Zn(II) ? Cu(I). Although the purified protein is unable to overcome the large thermodynamic preference for Cu(I) and exclude Zn(II) chelation in the presence of Ni(II), in vivo studies reveal a Ni(II)-specific function for the protein. Furthermore, these latter experiments support a specific role for SlyD as a [NiFe]-hydrogenase enzyme maturation factor. The implications of the divergence between the metal selectivity of SlyD in vitro and the specific activity in vivo are discussed.     

The copper-enzyme dopamine beta-monooxygenase: studies on the ability of several metals to inhibit the enzyme activity and to replace the copper

The ability of several metals to inhibit dopamine beta-monooxygenase was measured and compared with their ability to compete with the binding of 64Cu to the water-soluble form of the bovine adrenal enzyme at pH 6.0. In the presence of an optimal concentration of copper (0.5 microM in the present assay system), an inhibition was observed upon addition of Hg(II), Zn(II), or Ni(II). Only a small fraction of the inhibition with these metals may be due to uncoupling of electron transport from hydroxylation. Preincubation of these metals with the Cu-depleted apoenzyme before addition of copper, revealed a stronger inhibition than if copper was added before the other metals. Hg(II), Zn(II), and Ni(II) also compete with the binding of 64Cu(II) to the protein. Hg(II) was the most effective and Ni(II) the least effective of these metals, both with respect to inhibition of the enzyme activity and to prevent the binding of 64Cu(II). Competition experiments on the binding of Zn(II) and 64Cu in the presence and absence of ascorbate, indicated i) a similar affinity of Cu(I) and Cu(II) to the native enzyme, and ii) a more rapid binding of Cu(I) than Cu(II) to the Cu-depleted and Zn-containing enzyme. Al(III), Fe(II), Mg(II), Mn(II), Co(II), Cd(II), and Pb(II) neither inhibited the enzyme activity nor competed with the binding of 64Cu(II) to the protein (Fe(II) was not tested for binding). Of those metals cited above only Cu(II)/Cu(I) was able to reactivate the apoenzyme.     

Divalent metal derivatives of the hamster dihydroorotase domain.

Dihydroorotase (DHOase, EC 3.5.2.3) is a zinc enzyme that catalyzes the reversible cyclization of N-carbamyl-L-aspartate to L-dihydroorotate in the third reaction of the de novo pathway for biosynthesis of pyrimidine nucleotides. The recombinant hamster DHOase domain from the trifunctional protein, CAD, was overexpressed in Escherichia coli and purified. The DHOase domain contained one bound zinc atom at the active site which was removed by dialysis against the chelator, pyridine-2,6-dicarboxylate, at pH 6.0. The apoenzyme was reconstituted with different divalent cations at pH 7.4. Co(II)-, Zn(II)-, Mn(II)-, and Cd(II)-substituted DHOases had enzymic activity, but replacement with Ni(2+), Cu(2+), Mg(2+), or Ca(2+) ions did not restore activity. Atomic absorption spectroscopy showed binding of one Co(II), Zn(II), Mn(II), Cd(II), Ni(II), or Cu(II) to the enzyme, while Mg(II) and Ca(II) were not bound. The maximal enzymic activities of the active, reconstituted DHOases were in the following order: Co(II) --> Zn(II) --> Mn(II) --> Cd(II). These metal substitutions had major effects upon values for V(max); effects upon the corresponding K(m) values were less pronounced. The pK(a) values of the Co(II)-, Mn(II)-, and Cd(II)-substituted enzymes derived from pH-rate profiles are similar to that of Zn(II)-DHOase, indicating that the derived pK(a) value of 6.56 obtained for Zn-DHOase is not due to ionization of an enzyme-metal aquo complex, but probably a histidine residue at the active site. The visible spectrum of Co(II)-substituted DHOase exhibits maxima at 520 and 570 nm with molar extinction coefficients of 195 and 210 M(-1) cm(-1), consistent with pentacoordination of Co(II) at the active site. The spectra at high and low pH are different, suggesting that the environment of the metal binding site is different at these pHs where the reverse and forward reactions, respectively, are favored.     

Effects of metal on the biochemical properties of Helicobacter pylori HypB, a maturation factor of [NiFe]-hydrogenase and urease

The biosyntheses of the [NiFe]-hydrogenase and urease enzymes in Helicobacter pylori require several accessory proteins for proper construction of the nickel-containing metallocenters. The hydrogenase accessory proteins HypA and HypB, a GTPase, have been implicated in the nickel delivery steps of both enzymes. In this study, the metal-binding properties of H. pylori HypB were characterized, and the effects of metal binding on the biochemical behavior of the protein were examined. The protein can bind stoichiometric amounts of Zn(II) or Ni(II), each with nanomolar affinity. Mutation of Cys106 and His107, which are located between two major GTPase motifs, results in undetectable Ni(II) binding, and the Zn(II) affinity is weakened by 2 orders of magnitude. These two residues are also required for the metal-dependent dimerization observed in the presence of Ni(II) but not Zn(II). The addition of metals to the protein has distinct impacts on GTPase activity, with zinc significantly reducing GTP hydrolysis to below detectable levels and nickel only slightly altering the k(cat) and K(m) of the reaction. The regulation of HypB activities by metal binding may contribute to the maturation of the nickel-containing enzymes.     

Fluorescence-based biosensing of zinc using carbonic anhydrase

Measurement of free zinc levels and imaging of zinc fluxes remains technically difficult due to low levels and the presence of interfering cations such as Mg and Ca. We have developed a series of fluorescent zinc indicators based on the superb sensitivity and selectivity of a protein, human apo-carbonic anhydrase II, for Zn(II). These indicators transduce the level of free zinc as changes in intensity, wavelength ratio, lifetime, and/or anisotropy; the latter three approaches permit quantitative imaging of zinc levels in the microscope. A unique attribute of sensors incorporating biological macromolecules as transducers is their capability for modification by site-directed mutagenesis. Thus we have produced variants of carbonic anhydrase with improved affinity for zinc, altered selectivity, and enhanced binding kinetics, all of which are difficult to modify in small molecule indicators.     

alpha-Lactalbumin: structure and function

Small milk protein alpha-lactalbumin (alpha-LA), a component of lactose synthase, is a simple model Ca(2+) binding protein, which does not belong to the EF-hand proteins, and a classical example of molten globule state. It has a strong Ca(2+) binding site, which binds Mg(2+), Mn(2+), Na(+), and K(+), and several distinct Zn(2+) binding sites. The binding of cations to the Ca(2+) site increases protein stability against action of heat and various denaturing agents, while the binding of Zn(2+) to the Ca(2+)-loaded protein decreases its stability. Functioning of alpha-LA requires its interactions with membranes, proteins, peptides and low molecular weight substrates and products. It was shown that these interactions are modulated by the binding of metal cations. Recently it was found that some folding variants of alpha-LA demonstrate bactericidal activity and some of them cause apoptosis of tumor cells.     

Binding of cadmium(II) and zinc(II) to human and dog serum albumins. An equilibrium dialysis and 113Cd-NMR study.

The binding of Cd(II) and Zn(II) to human serum albumin (HSA) and dog serum albumin (DSA) has been studied by equilibrium dialysis and 113Cd(II)-NMR techniques at physiological pH. Scatchard analysis of the equilibrium dialysis data indicate the presence of at least two classes of binding sites for Cd(II) and Zn(II). On analysis of the high-affinity class of sites, HSA is shown to bind 2.08 +/- 0.09 (log K = 5.3 +/- 0.6) and 1.07 +/- 0.12 (log K = 6.4 +/- 0.8) moles of Cd(II) and Zn(II) per mole of protein, respectively. DSA bound 2.02 +/- 0.19 (log K = 5.1 +/- 0.8), and 1.06 +/- 0.15 (log K = 6.0 +/- 0.2) moles of Cd(II) and Zn(II) per mole of protein, respectively. Competition studies indicate the presence of one high-affinity Cd(II) site on both HSA and DSA that is not affected by Zn(II) or Cu(II), and one high-affinity Zn(II) site on both HSA and DSA that is not affected by Cd(II) or Cu(II). 113Cadmium-HSA spectra display three resonances corresponding to three different sites of complexation. In site I, Cd(II) is most probably coordinated to two or three histidyl residues, site II to one histidyl residue and three oxygen ligands (carboxylate), while for the most upfield site III, four oxygens are likely to be involved in the binding of the metal ion. The 113Cd(II)-DSA spectra display only two resonances corresponding to two different sites of complexation. The environment around Cd(II) at sites I and II on DSA is similar to sites I and II, respectively, on HSA. No additional resonances are observed in any of these experiments and in particular in the low field region where sulfur coordination occurs. Overall, our results are consistent with the proposal that the physiologically important high-affinity Zn(II) and Cd(II) binding sites of albumins are located not at the Cu(II)-specific NH2-terminal site, but at internal sites, involving mostly nitrogen and oxygen ligands and no sulphur ligand.     

Effects of Zn(II) binding and apoprotein structural stability on the conformation change of designed antennafinger proteins

Ligand-induced conformation change is a general strategy for controlling protein function. In this work, we demonstrate the relationships between ligand binding and conformational stability using a previously designed protein, Ant-F, which undergoes a conformation change upon Zn(II) binding. To investigate the effect of stabilization of the apo structure on the conformation change, we also created a novel protein, Ant-F-H1, into which mutations are introduced to increase its stability over that of Ant-F. The chemical denaturation experiments clarified that apo-Ant-F-H1 is more stable than apo-Ant-F (DeltaDeltaG = -1.28 kcal/mol) and that the stability of holo-Ant-F-H1 is almost the same as that of holo-Ant-F. The Zn(II) binding assay shows that the affinity of Zn(II) for Ant-F-H1 is weaker than that for Ant-F (DeltaDeltaG = 1.40 kcal/mol). A large part of the increased value of free energy in stability corresponds to the decreased value of free energy in Zn(II) binding, indicating that the stability of the apo structure directly affects the conformation change. The denaturation experiments also reveal that Zn(II) destabilizes the conformation of both proteins. From the thermodynamic linkage, Zn(II) is thought to bind to the unfolded state with high affinity. These results suggest that the binding of Zn(II) to the unfolded state is an important factor in the conformational change as well as the stability of the apo and holo structures.     

The chromosomally encoded cation diffusion facilitator proteins DmeF and FieF from Wautersia metallidurans CH34 are transporters of broad metal specificity

Genomic sequencing of the beta-proteobacterium Wautersia (previously Ralstonia) metallidurans CH34 revealed the presence of three genes encoding proteins of the cation diffusion facilitator (CDF) family. One, CzcD, was previously found to be part of the high-level metal resistance system Czc that mediates the efflux of Co(II), Zn(II), and Cd(II) ions catalyzed by the CzcCBA cation-proton antiporter. The second CDF protein, FieF, is probably mainly a ferrous iron detoxifying protein but also mediated some resistance against other divalent metal cations such as Zn(II), Co(II), Cd(II), and Ni(II) in W. metallidurans or Escherichia coli. The third CDF protein, DmeF, showed the same substrate spectrum as FieF, but with different preferences. DmeF plays the central role in cobalt homeostasis in W. metallidurans, and a disruption of dmeF rendered the high-level metal cation resistance systems Czc and Cnr ineffective against Co(II). This is evidence for the periplasmic detoxification of substrates by RND transporters of the heavy metal efflux family subgroup.     

Alpha-sarcin cleavage of ribosomal RNA is inhibited by the binding of elongation factor G or thiostrepton to the ribosome.

The translocation reaction catalyzed by elongation factor G (EF-G) is inhibited either by alpha-sarcin cleavage of 23S rRNA or by the binding of thiostrepton to the E. coli ribosome. Here we show that the transitory binding of EF-G and GDP to the ribosome inhibited the rate of alpha-sarcin cleavage and that stabilization of this binding with fusidic acid completely prevented alpha-sarcin cleavage. A similar pattern of inhibition was seen upon the binding of elongation factor 2 to the S. cerevisiae ribosome. The irreversible binding of the antibiotic thiostrepton to the E. coli ribosome, on the other hand, decreased the rate of cleavage by alpha-sarcin approximately 2-fold. These results suggest that the alpha-sarcin site is located within the ribosomal domain for EF-G binding and that the conformation of this site is affected by the binding of thiostrepton.     

Fusion of phospholipid vesicles produced by the anti-tumour protein alpha-sarcin.

The anti-tumour protein alpha-sarcin causes fusion of bilayers of phospholipid vesicles at neutral pH. This is demonstrated by measuring the decrease in the efficiency of the fluorescence energy transfer between N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-dimyristoylphosphatidylethano lamine (NDB-PE) (donor) and N-(lissamine rhodamine B sulphonyl)-diacylphosphatidylethanolamine (Rh-PE) (acceptor) incorporated in dimyristoylphosphatidylcholine (DMPG) vesicles. The effect of alpha-sarcin is a maximum at 0.15 M ionic strength and is abolished at basic pH. alpha-Sarcin promotes fusion between 1,6-diphenylhexa-1,3,5-triene (DPH)-labelled DMPG and dipalmitoyl-PG (DPPG) vesicles, resulting in a single thermotropic transition for the population of fused phospholipid vesicles. Bilayers composed of DMPC and DMPG, at different molar ratios in the range 1:1 to 1:10 PC/PG, are also fused by alpha-sarcin. Freeze-fracture electron micrographs corroborate the occurrence of fusion induced by the protein. alpha-Sarcin also modifies the permeability of the bilayers, causing the leakage of calcein in dye-trapped PG vesicles. All of the observed effects reach saturation at a 50:1 phospholipid/protein molar ratio, which is coincident with the binding stoichiometry previously described.