Release of the neocarzinostatin chromophore from the holoprotein does not require major conformational change of the tertiary and secondary structures induced by trifluoroethanol

Neocarzinostatin is a potent enediyne antitumor antibiotic complex in which a chromophore is noncovalently bound to a carrier protein. The protein regulates availability of the drug by proper release of the biologically active chromophore. To understand the physiological mechanism of the drug delivery system, we have examined the trifluoroethanol (TFE)-induced conformational changes of the protein with special emphasis on their relation to the release of the chromophore from holoneocarzinostatin. The effect of the alpha helix-inducing agent, TFE, on all the beta-sheet neocarzinostatin proteins was studied by circular dichroism, fluorescence, and (1)H NMR studies. By using binding of anilinonaphthalene sulfonic acid as a probe, we observed that the protein exists in a stable, partially structured intermediate state around 45-50% TFE, which is consistent with the results from tryptophan fluorescence and circular dichroism studies. The native state is stable until 20% TFE and is half-converted into the intermediate state at 30% TFE, which starts to collapse beyond 50%. High pressure liquid chromatographic analysis of the release of the chromophore caused by TFE treatment at 0 degrees C suggests that the release process, which occurs below 20% TFE, does not result from an observable conformational change in the protein. Kinetic measurements of the release of chromophore at 25 degrees C reveal that TFE does stimulate the rate of release, which increases sharply at 15% and reaches a maximum at 20% TFE, although no major secondary or tertiary structural change of the carrier protein is observed under these same conditions. Our data suggest that chromophore release results from a fluctuation of the protein structure that is stimulated by TFE. Complete release of the chromophore occurs at TFE concentrations where no overall observable unfolding of the apoprotein is seen. Thus, the results suggest that denaturation of the protein by TFE is not a necessary step for release of the tightly bound chromophore.     

Circular dichroism of metallothioneins. A structural approach.

A comprehensive study on circular dichroism of metallothioneins containing Zn, Cd and Cu was carried out. The contributions of the metals, the sulphur and the polypeptide chain to the observed Cotton effects was shown. From the pH dependency of the extrinsic Cotton effects which are due to the metal-thiolate chromophore the stability of the metal clusters was found to decrease in the order Cu greater than Cd greater than Zn. The pH values corresponding to the dissociation of half of the bound metal ions are 0.44 for Cu-thionein, 3.05 for Cd-thionein and 4.6 for Zn-thionein. The extrinsic Cotton effects of Cd, Zn-thioneins of varying Cd to Zn ratio could be simulated using the difference circular dichroic spectra of Cd-thionein (bands at 227, 242.5 and 262 nm), Zn-thionein (bands at 225 and 244 nm) and the circular dichroic spectrum of cysteine-thionein (band at 200 nm, shoulder at 225 nm). Since during the dissociation of the metals the circular dichroic spectra exhibited changes only in amplitude and not in shape we can conclude that the dissociation of the metal ions involves the complete sequential degradation of metal clusters. In the near-ultraviolet region the metal-free proteins show only Cotton effects attributable to a disulphide chromophore. Thus Cotton bands are observed for cystine-thionein at 282.5 and 260 nm. From the intrinsic circular dichroism of Cd- and Zn-thionein (negative Cotton effect at 200 nm, shoulder at 225 nm) it follows that the protein conformation consists of less than 5% helical or pleated sheet structure and therefore has to be classified as unordered structure or "fixed" random coil     

Differences in the circular dichroic spectra of various species serum albumin complexes with novobiocin

The circular dichroic spectra of 8 different species albumin-novobiocin complexes appear to be unique. In the spectral region of interest a chromophore of the novobiocin is perturbed by the complexing with the albumin. Each species albumin imparts a different amount of perturbation which results in a unique circular dichroic spectrum. At least a part of this perturbation is a conformational change induced in the drug.     

Self-association of apo-C-I from the human high density lipoprotein complex.

The molecular properties of apo-C-I, isolated from the human high density lipoprotein complex, have been evaluated as a function of pH, solvent composition, and protein concentration by sedimentation equilibrium and circular dichroic measurements. This protein self-associates in aqueous solution at neutral pH with concomitant changes in secondary structure. In contrast, in the acid pH range, apo-C-I is monomeric and its ellipticity is independent of protein concentration. The results are discussed in terms of the interpretation of experiments where changes in the physical properties of apolipoproteins have been used to monitor ligand binding and lipid-apolipoprotein recombination.     

A new model for ligand release. Role of side chain in gating the enediyne antibiotic

Antitumor antibiotic chromoproteins such as neocarzinostatin involve a labile toxin that is tightly bound by a protective protein with very high affinity but must also be freed to exert its function. Contrary to the prevalent concept of ligand release, we established that toxin release from neocarzinostatin requires no major backbone conformational changes. We report, herein, that subtle changes in the side chains of specific amino acid residues are adequate to gate the release of chromophore. A recombinant wild type aponeocarzinostatin and its variants mutated around the opening of the chromophore binding cleft are employed to identify specific side chains likely to affect chromophore release. Preliminary, biophysical characterization of mutant apoproteins by circular dichroism and thermal denaturation indicate that the fundamental structural characteristics of wild type protein are conserved in these mutants. The chromophore reconstitution studies further show that all mutants are able to bind chromophore efficiently with similar complex structures. NMR studies on 15N-labeled mutants also suggest the intactness of binding pocket structure. Kinetic studies of chromophore release monitored by time course fluorescence and quantitative high pressure liquid chromatography analyses show that the ligand release rate is significantly enhanced only in Phe78 mutants. The extent of DNA cleavage in vitro corresponds well to the rate of chromophore release. The results provide the first clear-cut indication of how toxin release can be controlled by a specific side chain of a carrier protein.     

Neocarzinostatin chromophore binds to deoxyribonucleic acid by intercalation

The nonprotein chromophore of neocarzinostatin was found to share many of the characteristics of classical intercalators in its interaction with DNA. Viscosity studies with PM2 DNA indicated that the DNA helix unwinding induced by the chromophore was 0.82 times that of ethidium or 21 degrees. Electric dichroism of the chromophore--DNA complex showed that each bound chromophore molecule lengthened DNA by 3.3 A and that absorbance transitions of the chromophore at 315--385 nm were oriented approximately parallel to DNA bases, as expected for an intercalated aromatic ring. Binding to DNA induced strong hypochromicity and a pronounced red shift in the absorbance spectrum of the chromophore. Spectrophotometric titrations suggested at least two types of chromophore binding sites on DNA; one type of site was saturated at rb = 0.125 chromophore molecule/nucleotide, but binding to additional sites continued to at least rb = 0.3. These physical--chemical studies were performed at pH 4--5 in order to keep the chromophore stable, but chromophore bound to an excess of DNA at pH 7 showed a stable absorbance spectrum identical with that seen at pH 4--5, suggesting that a similar type of binding occurs at neutral pH. Chromophore which had spontaneously degraded in pH 8 buffer did not bind to DNA at all, as judged by absorbance spectroscopy. The degree of protection afforded by DNA against spontaneous chromophore degradation implied a dissociation constant of approximately 5 microM for the DNA--chromophore complex at neutral pH and physiological ionic strength. Supercoiled DNA was nearly twice as effective as relaxed DNA in protecting chromophore from degradation, providing additional evidence for intercalation at neutral pH. Comparison of absorbance, fluorescence, and dichroism spectra suggests that the naphthalene ring system is the intercalating moiety.     

Generation of superoxide free radical by neocarzinostatin and its possible role in DNA damage

Spectroscopic analysis of the reduction of both nitro blue tetrazolium and ferricytochrome c induced by neocarzinostatin shows that superoxide free radical is produced during the spontaneous degradation of the antibiotic. The amount of superoxide free radical produced from neocarzinostatin is not affected by the presence of thiol, although earlier work has shown that DNA damage is stimulated at least 1000-fold by thiol. Transition metals are not involved in this reaction. Although superoxide dismutase inhibits the reduction of nitro blue tetrazolium and cytochrome c induced by neocarzinostatin, neither it nor catalase interferes with the action of neocarzinostatin on DNA, whether or not drug has been activated by thiol. The pH profiles for spontaneous base release and alkali-labile base release (a measure of nucleoside 5'-aldehyde formation at a strand break) do not correspond with that for the generation of superoxide free radical from neocarzinostatin. The same holds for supercoiled DNA cutting by neocarzinostatin chromophore in the absence of a thiol, which is an acid-favored reaction. These results indicate that the generation of superoxide free radical by the drug does not correlate with DNA damage activity, whether or not thiol is present. Furthermore, the failure of hydroxyl free-radical scavengers to inhibit drug-induced single-strand breaks in supercoiled DNA in the absence of thiol also indicates that a diffusible hydroxyl free radical is most probably not involved in this reaction.     

Degradation of HeLa cell chromatin by neocarzinostatin and its chromophore

Chromatin is the in vivo target site for neocarzinostatin, a DNA strand scission antitumor drug. The effect of neocarzinostatin and its active chromophore component on HeLa cell chromatin is described here. Chromatin consisting of a mixture of mono-, di-, tri- and larger nucleosome fragments is prepared by micrococcal nuclease digestion of HeLa cell nuclei. Drug-induced conversion of chromatin to smaller sized fragments is measured by electrophoresis of the DNA on non-denaturing 4% polyacrylamide gels. Chromatin breakdown measured under these conditions is double-stranded in nature. In the presence of 2 mM dithiothreitol, neocarzinostatin causes degradation of large chromatin fragments and a loss of distinct nucleosome peaks. Detection of chromatin breakdown by neocarzinostatin is dependent upon the concentration of chromatin in the assay. When chromatin is increased from 14 to 70 micrograms/ml, changes in the larger fragments caused by 100 micrograms/ml neocarzinostatin become less obvious are are almost undetectable at 140 micrograms/ml chromatin. No change is observed when chromatin is treated with either neocarzinostatin or its chromophore in the absence of dithiothreitol. For detectable levels of chromatin degradation, 10 micrograms/ml neocarzinostatin is required compared to only 2.5 microgram/ml chromosome (expressed in microgram equivalent neocarzinostatin). Such degradation also occurs more rapidly with chromophore than with neocarzinostatin. Digestion of chromatin with neocarzinostatin continues for at least 30 min at 37 degrees C, while similar degradation caused by chromophore is complete in 1 min. Neocarzinostatin levels which actively degrade isolated chromatin can also effect release of soluble chromatin from intact nuclei. The released chromatin can serve as a substrate for micrococcal nuclease digestion. Such chromatin studies should prove useful in characterizing the mechanism of action of DNA reactive drugs such as neocarzinostatin.     

Free radical mechanisms in neocarzinostatin-induced DNA damage

The molecular mechanisms by which the antitumor protein antibiotic, neocarzinostatin, interacts with DNA and causes DNA sugar damage is discussed. Physical binding of the nonprotein chromophore of neocarzinostatin to DNA, involving an intercalative process and dependent on the microheterogeneity of DNA structure, is followed by thiol activation of the drug to a probable radical species. The latter attacks the deoxyribose, especially at thymidylate residues, by abstracting a hydrogen atom from C-5' to generate a carbon-centered radical on the DNA. This nascent form of DNA damage either reacts with dioxygen to form a peroxyl radical derivative, which eventuates in a strand break with a nucleoside 5'-aldehyde at the 5'-end or reacts with the bound drug to form a novel drug-deoxyribose covalent adduct. Nitroaromatic radiation sensitizers can substitute for dioxygen, but the DNA damage products are different. Similarities between the various biological effects of neocarzinostatin and ionizing radiation are reviewed.     

The inhibitory mechanism of in vitro protein phosphorylation by a nonprotein chromophore removed from neocarzinostatin

The inhibitory effect of a nonprotein chromophore removed from neocarzinostatin on protein phosphorylation by nuclear protein kinase in vitro has been studied. Low levels of the chromophore greatly inhibited protein phosphorylation in vitro. This inhibition, however, was not selectively dependent on the indicated kinases and their different phosphate acceptors (histones and non-histone protein). In contrast, the protein component (apoprotein) of neocarzinostatin did not affect the phosphorylation even at a concentration of 400-times higher than that of the chromophore. Moreover, apoprotein suppressed the chromophore-induced inhibition of protein phosphorylation in vitro in proportion to the apoprotein concentrations. Kinetic and analytical experiments suggest that the chromophore-induced inhibition of protein phosphorylation seems to be due to the binding of the chromophore to the kinases. In addition, we found that ultraviolet irradiation as well as methanol extraction can release the chromophore from neocarzinostatin, but it exhibits no inhibitory activity of DNA synthesis in growing cells. The fact that the chromophore-induced inhibition of protein phosphorylation in vitro was not sensitive to ultraviolet irradiation, which rapidly inactivated the ability of the chromophore to induce DNA degradation in vitro, suggests that there are different actions involved in the two inhibitions induced by the chromophore which is removed from neocarzinostatin.     

Regiospecific O-methylation of naphthoic acids catalyzed by NcsB1, an O-methyltransferase involved in the biosynthesis of the enediyne antitumor antibiotic neocarzinostatin

Neocarzinostatin, a clinical anticancer drug, is the archetypal member of the chromoprotein family of enediyne antitumor antibiotics that are composed of a nonprotein chromophore and an apoprotein. The neocarzinostatin chromophore consists of a nine-membered enediyne core, a deoxyaminosugar, and a naphthoic acid moiety. We have previously cloned and sequenced the neocarzinostatin biosynthetic gene cluster and proposed that the biosynthesis of the naphthoic acid moiety and its incorporation into the neocarzinostatin chromophore are catalyzed by five enzymes NcsB, NcsB1, NcsB2, NcsB3, and NcsB4. Here we report the biochemical characterization of NcsB1, unveiling that: (i) NcsB1 is an S-adenosyl-L-methionine-dependent O-methyltransferase; (ii) NcsB1 catalyzes regiospecific methylation at the 7-hydroxy group of its native substrate, 2,7-dihydroxy-5-methyl-1-naphthoic acid; (iii) NcsB1 also recognizes other dihydroxynaphthoic acids as substrates and catalyzes regiospecific O-methylation; and (iv) the carboxylate and its ortho-hydroxy groups of the substrate appear to be crucial for NcsB1 substrate recognition and binding, and O-methylation takes place only at the free hydroxy group of these dihydroxynaphthoic acids. These findings establish that NcsB1 catalyzes the third step in the biosynthesis of the naphthoic acid moiety of the neocarzinostatin chromophore and further support the early proposal for the biosynthesis of the naphthoic acid and its incorporation into the neocarzinostatin chromophore with free naphthoic acids serving as intermediates. NcsB1 represents another opportunity that can now be exploited to produce novel neocarzinostatin analogs by engineering neocarzinostatin biosynthesis or applying directed biosynthesis strategies.     

Stabilization of neocarzinostatin nonprotein chromophore activity by interaction with apoprotein and with HeLa cells

The methanol-extracted, nonprotein chromophore of the protein antibiotic neocarzinostatin (NCS), which possesses the full in vitro and in vivo deoxyribonucleic acid (DNA) strand-breaking activities and the ability to inhibit DNA synthesis and growth in HeLa cells of the holoantibiotic, is much more labile to inactivation by heat, 2-mercaptoethanol, long-wavelength UV light, and pH values above 4.8. Inactivation is inversely related to the methanol concentration. The pH activity profile of the isolated chromophore extends to pH values below 7.0. Chromophore inactivation is specifically blocked by the apoprotein of NCS; 100-fold higher concentrations of the apoprotein of another protein antibiotic, auromomycin, gave similar protection, whereas bovine serum albumin is even less effective. The chromophore, and not the apoprotein, is inactivated by heat or light (360 nm) as determined by both activity and isoelectric focusing experiments. In contrast to other chromophoric antibiotic substances (daunorubicin and the extracted chromophore of aurodomomycin), the NCS chromophore interacts irreversibly with HeLa cells at 0 degrees C in serum-free medium so as to inhibit subsequent DNA synthesis at 37 degrees C. Such interaction at 0 degrees C is very rapid, reaching 50% completion in about 15 s, and is not found with native NCS or when apo-NCS is added before the chromophore or when serum is included in the preincubation at 0 degrees C. Washing with apo-NCS or serum-containing (or-free) medium after preincubation of the cells with the chromophore at 0 degrees C fails to reverse the subsequenct inhibition of DNA synthesis.     

A synthetic peptide encompassing the binding site of the second zinc atom (the 'structural' zinc) of alcohol dehydrogenase.

A 23-residue peptide was synthesized that incorporates the loop which binds the structural zinc atom of mammalian alcohol dehydrogenases and contributes, in part, to subunit interactions in the native enzyme. Neither the amino acid composition nor the sequence of the peptide resemble those of zinc fingers. The reduced peptide stoichiometrically binds zinc or cobalt to form stable complexes with a dissociation constant of the peptide/CO2+ complex of 2.1 microM at pH 7.5. EDTA disrupts the complex. The absorption and magnetic circular dichroic spectra of the cobalt-peptide are indicative of a tetrahedral coordination geometry, and are similar to those of the cobalt-substituted structural site of horse and human (beta 1 beta 1) liver alcohol dehydrogenases. Consequently, the synthetic peptide can serve as a model for the metal-binding segment of alcohol dehydrogenase and for studies of fundamental problems concerning protein/metal interactions.     

Incorporation of 18O2 into thymidine 5'-aldehyde in neocarzinostatin chromophore-damaged DNA

Strand scission of DNA by the chromophore of neocarzinostatin converts the 5'-hydroxyl of deoxyribose to a 5'-aldehyde. The origin of the aldehydic oxygen has now been elucidated by mass spectrometry. DNA-associated thymidine 5'-aldehyde produced by treatment of DNA with neocarzinostatin chromophore in 2H218O/16O2 or in 2H216O/18O2 was reduced, liberated by nuclease treatment, permethylated, and analyzed by gas chromatography-mass spectrometry. The data clearly show that molecular oxygen is the only source of the 5'-aldehydic oxygen. The addition of molecular oxygen at C-5', possibly via a reactive form of neocarzinostatin chromophore, must be involved; a carbonium ion intermediate at C-5' is ruled out.     

Optical properties of hyaluronic acid. Ultraviolet circular dichroism and optical rotatory dispersion

The molar optical rotation at 220 nm and ellipticity values at 210 nm of both sodium hyaluronate and hyaluronic acid are greatly enhanced in comparison to the values for the monomeric units and oligosaccharides indicating a degree of preferred order. With increasing hydrogen ion concentration, there is no appreciable change in the 210 nm circular dichroic band, but the second circular dichroic band below pH 4 changes abruptly to the positive side and reaches a maximum value at pH 2·5. This positive circular dichroic band of hyaluronic acid is temperature and concentration dependent. The major change in sign and position of the second circular dichroic band of hyaluronic acid below pH 4 is attributed to the conformational change of a single polysaccharide chain or to a chain-chain interaction. The results indicate that increase in concentration or decrease in temperature and in the ionization of carboxyl group promotes the formation of ordered cross-link regions. The conformational changes found in solution have been interpreted as an order-disorder transition in the crosslink regions based on the interconversion of random coil and double helix.     

The macromolecular properties of peanut agglutinin

The dependence upon solution conditions of the quaternary structure and gross conformation of peanut agglutinin was examined by sedimentation equilibrium, sedimentation velocity, gel chromatography, and circular dichroism. At pH 8, the protein exists as a compactly folded tetramer of molecular weight 98,000. Between pH 4.75 and pH 3.0, the molecular reversibly dissociates to a (still globular) dimer. In the presence of denaturants such as SDS or guanidinium chloride, the protein dissociates to its four equal-sized constituents polypeptide chains. The circular dichroic spectrum of peanut lectin exhibits changes in the near ultraviolet upon binding of lactose, whereas the far ultraviolet spectrum remains unchanged. Dissociation to the dimeric state produces subtle changes in both the near and far ultraviolet circular dichroic spectrum.     

Neocarzinostatin: interaction between the antitumor-active chromophore and the carrier protein.

Conformational analysis of neocarzinostatin, an antibiotic protein with antitumor activity, in its holo-state in solution was carried out by NMR-spectroscopy. The results showed a beta-barrel structure for the carrier protein, in which the chromophore is tightly enwrapped and thus shielded from exposure to the solvent. The three-dimensional structure of this complex led to the proposal of a possible mechanism for the exposure or release of the active principle due to subtle environmental changes, e.g., in pH.     

The generalization of pre-neocarzinostatin and antagonism of neocarzinostatin-induced DNA scission in vitro

Pretreatment of the antitumor protein neocarzinostatin with heat, ultraviolet or white light, and thiols inactivates the drug, as measured by the cessation of phage T2 DNA strand scission in vitro. The inactive forms obtained are identical with pre-neocarzinostatin on the basis of isoelectric focusing, molecular weight determination, and changes in circular dichroism spectra. In incubations together with neocarzinostatin and T2 DNA, the inactive form inhibits strand scission to a considerable degree. This result suggests that both forms compete for a limited number of available DNA binding sites.     

On the various types of circular dichroism induced on acridine orange bound to poly(S-carboxymethyl-L-cysteine).

Circular dichroism and absorption spectra are measured on mixed solutions of acridine orange and poly(S-carboxymethyl-L -cysteine) at different pH and P/D mixing ratios. The observed circular dichroism spectra are classified into several types, mainly based on the number and sign of circular dichroic bands in the visible region. Three of them are associated with the absorption spectra characteristic of dimeric dye or higher aggregates of dye. Type I is observed with solutions, of which the pH is acid and P/D is higher than 4, and it has an unsymmetrical pair of positive and negative dichroic bands at 470 and 430 nm. This type is induced on the dye bound to the polymer in the β-conformation. Types II and III are considered to be characteristic of randomly coiled polymers. Type II is exhibited by solutions of P/D higher than 1 at pH 5–7 and has two dichroic bands around the same wavelengths as Type I but with opposite signs and an additional positive band at 560 nm. Type III, shown by solutions of P/D 2–0.6 at pH 6–10.5, has three dichroic bands around the same wavelengths as Type II but with signs opposite to it. The other two types of circular dichroism, induced for the solutions of P/D less than 1 at slightly acid pH, are associated with the absorption spectra of monomeric dye and are observed with disordered or randomly coiled polymer. They have a pair of dichroic bands at 540 and 425 nm, and the signs of these bands are opposite to each other in these two types.     

Molecular dynamics simulations investigation of neocarzinostatin chromophore-releasing pathways from the holo-NCS protein

The enediyne ring chromophore with strong DNA cleavage activity of neocarzinostatin is labile and therefore stabilization by forming the complex (carrying protein + chromophore: holo-NCS). Holo-NCS has gained much attention in clinical use as well as for drug delivery systems, but the chromophore-releasing mechanism to trigger binding to the target DNA with high affinity and producing DNA damage remain unclear. Three possible pathways were initially determined by conventional MD, essential dynamics and essential dynamics sampling. One of the paths runs along the naphthoate moiety; another runs along the amino sugar moiety; the third along the enediyne ring. Further, calculated forces and time by FPMD (force-probe molecular dynamics) suggest that the opening of the naphthoate moiety is most favorable pathway and Leu45, Phe76 and Phe78 all are key residues for chromophore release. In addition, conformational analyses indicate that the chromophore release is only local motions for the protein.