©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Different Conformations and Site Selectivity of HO-Co(III)-Bleomycin A and Co(III)-Bleomycin A Bound to DNA Oligomers (*)

(Received for publication, September 21, 1995; and in revised form, December 13, 1995)

Qunkai Mao Patricia Fulmer Wenbao Li Eugene F. DeRose David H. Petering (§)

From the Department of Chemistry, University of Wisconsin, Milwaukee, Wisconsin 53201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Conformational properties of HO(2)-Co(III)-bleomycin A(2) (Form I) and Co(III)-bleomycin (Form II) bound to DNA oligomers offering either principal cleavage site for the drug, d(GGAAGCTTCC)(2) or d(AAACGTTT)(2), have been studied by NMR methods. Form I binds in slow exchange to these oligomers. It retains most of its solution nuclear Overhauser effects (NOEs) upon binding to either oligomer. Pyrimidinyl methyl protons from the metal domain of the drug make an NOE connection with a G5 2-amino proton on DNA. The bithiazole intercalates between base pairs involving either C6 and T7 or T6 and T7 of the two DNA molecules, according to NOE connections between the bithiazole protons and protons from these bases and changes in the positions of their chemical shifts. Form II also retains most of its solution NOEs upon association with the first oligomer. However, in contrast to Form I it binds to DNA in fast exchange on the NMR time scale over the temperature range of 5-35 °C and does not break the degeneracy of the DNA proton chemical shifts. No intermolecular NOEs between Form II and the 10-mer have been detected. Likewise, the major perturbation in chemical shift of the histidine H2 and guanine G5 protons seen in Form I-DNA adducts is absent in Form II-DNA. The association constant of Form II with d(GGAAGCTTCC)(2) in 20 mM HEPES buffer at pH 7.4 and 25 °C is 1.7 times 10^5M, and 1.0 mol of Form II bind per mol of 10-mer.


INTRODUCTION

Bleomycin (Blm) (^1)is an antitumor agent used to treat human cancer. It is comprised of metal- and DNA-binding domains linked by a peptide and a disaccharide unit (Fig. 1). Fe(II)-Blm reacts with O(2) and reductants to generate HO(2)-Fe(III)-Blm, which initiates single and double strand DNA cleavage and base release (Burger et al., 1981; Petering et al., 1990; Sam et al., 1994; Stubbe and Kozarich, 1987). Efficient reaction is due, in part, to the binding of the drug to DNA through the bithiazole and positively charged R group (Chien et al., 1977). There is definite site selectivity for the reactions of Fe-Blm with DNA to produce strand cleavage or base release. The drug prefers initial attack on the deoxyribose unit of the sequences 5`-GpT-3` and 5`-GpC-3` (McLean et al., 1989; Steighner and Povirk, 1990).


Figure 1: Structure of bleomycin. Only the deprotonated amide nitrogen contributes to the ligand charge among the metal binding sites (dotted). It is also assumed that the peroxyl group is protonated.



The redox chemistry of Fe-Blm in solution or bound to DNA can be modeled closely by Co-Blm (Xu et al., 1992a, 1992b). Both form dioxygenated complexes, which undergo dimerization to reach a peroxyl species, HO(2)-Co(III)-Blm (Form I) and Co(III)-Blm (Form II) or the corresponding iron species. However, in contrast to HO(2)-Fe(III)-Blm, its cobalt analog is stable in the presence of DNA.

The three-dimensional conformations of the two Co-Blm structures have been determined by NMR spectroscopy (Xu et al., 1994). Both forms display extensively folded structures in which the peptide linker is close-packed against the metal domain. In addition, in Form I the bithiazole moiety is folded back over the metal domain. These structures also bind tightly to DNA, offering the opportunity to examine them complexed with DNA as models for the related Fe-Blms (Xu et al., 1992b).

Recent communications have begun to reveal how metallobleomycins bind to DNA. NMR data on the interaction of Form I with DNA 10-mer, d(CCAGGCCTGG)(2) showed that the complex binds in slow exchange with DNA and that the bithiazole moiety partially intercalated into the base pair structure between C6 and C7 directly adjacent to the GpC cleavage site (Wu et al., 1994). Based on provisional NOE assignments, it was proposed that the pyrimidine methyl group of Form I was in the proximity of a 2-amino proton of G5 at the site. Another study on the interaction of Zn-Blm with DNA concluded that Blm binds in the minor groove in rapid exchange with oligomeric DNA (Manderville et al., 1994; Manderville et al., 1995).

The finding that Form I and Zn-Blm bind differently to DNA oligomers despite containing the same linker and DNA binding domains suggests that metal domain properties play a key role in the binding process. From NMR studies and associated molecular dynamic calculations, it appears that the metal domains of Forms I and II Co-Blm bind Co(III) with opposite chiralities (Xu et al., 1994). Therefore, it was hypothesized that Forms I and II should associate differently with DNA. The present study compares the interaction of Forms I and II with a DNA 10-mer containing a GpC site to test this hypothesis. It also examines the binding of Form I to another DNA oligomer bearing a GpT site to determine whether the features of binding of Form I to DNA are common to different preferential binding sites.


EXPERIMENTAL PROCEDURES

Preparation of DNA Oligomers

Syntheses of d(GGAAGCTTCC) and d(AAACGTTT) were accomplished using a Millipore Cyclone Plus DNA synthesizer and nucleotide amidites from Millipore as starting materials. The method followed directions provided by the manufacturer except as modified below (Millipore, 1989). After synthesis of 30 mmol of either oligomer, each was cleaved from the support and fully deprotected by incubation for 48 h in 30% ammonium hydroxide. Purification of the oligomers was done with a Millipore C8 reverse phase column (250 times 4.6 mm). Solvent A contained 0.1 M sodium acetate, pH 6.5, and 5% acetonitrile. Solvent B was a mixture of 0.1 M sodium acetate and 65% acetonitrile. The gradient began with 100% solvent A and reached 100% solvent B in 45 min with a flow rate of 3.0 ml/min. Finally, to remove trityl groups, 6 ml of 80% acetic acid was incubated for 1 h with oligomer. To separate the dimethoxytrityl groups from the oligomer, a Sephadex G-15 desalting column was used with 20 mM ammonium bicarbonate, pH 7.4, as the eluate buffer.

Preparation of Form I and Form II and Their DNA Adducts

Blenoxane, the clinical mixture primarily of Blm A(2) and B(2), was a gift of Bristol Myers Co. Bleomycin A(2) was isolated from Blenoxane as described previously (Xu et al., 1994). Then, a one-to-one mixture of Form I and Form II was made by reacting Blm A(2) with Co(II)Cl(2) with stirring under aerobic conditions. The mixture was separated by high pressure liquid chromatography using a 250 times 4.6-mm C18 reverse phase semipreparative column (Bio-Rad) with the following gradient. Solvent A contained water and 0.2% (v/v) trifluoroacetic acid. Solvent B was methanol. The gradient changed linearly from 70% solvent A and 30% solvent B to 63% solvent A and 37% solvent B within 10 min at a flow rate of 2.0 ml/min. When 200-µl fractions were collected, two peaks appeared in the chromatogram representing Form II and Form I with characteristic retention times of 7.8 and 8.9 min, respectively. In order to remove trifluoroacetic acid and methanol from the isolated Form I or Form II mixtures, the samples were lyophilized several times in water. The Form I or Form II DNA adducts were made by mixing isolated Co-Blm species with DNAa or DNAb.

NMR Spectroscopy

Proton NMR spectra were acquired at 500 MHz on a GE GN500 NMR spectrometer. Phase-sensitive two-dimensional spectra were acquired using TPPI-States phase cycling (Marion et al., 1989). NOESY spectra were acquired in D(2)O with a mixing time of 250 ms at 15 and 25 °C (Jeener, et al., 1979; Macura et al., 1981). TOCSY spectra were acquired in D(2)O at 15 °C with a mixing time of 80 ms using a WALTZ-17(y) mixing scheme (Bax, 1989; Braunschweiler and Ernst, 1983). The proton sweep widths in the D(2)O spectra were set to 6024 Hz, centered on the water resonance, with 2048 complex points acquired in t(2), and 256 complex t(1) increments collected, corresponding to acquisition times of 340 and 42 ms in the two dimensions. The residual HOD peak was suppressed with an on resonance DANTE pulse sequence applied during the second half of the 1-s recovery delay between scans. NOESY spectra in 90% H(2)O, 10% D(2)O were acquired with mixing times of 200 ms at 5 and 15 °C. The water resonance was suppressed using a ``jump and return'' read pulse, with an 80-µs delay between pulses, corresponding to excitation maxima of ± 3125 Hz from the carrier frequency centered on the water resonance (Plateau and Gueron, 1982). A 45° phase shift was added to the normal mixing pulse-phase cycling scheme to minimize radiation damping (Driscoll et al., 1989). The proton sweep widths were set to 12,048 Hz, with 2048 complex points acquired in t(2), and 256 complex t(1) increments collected, corresponding to acquisition times of 170 and 21 ms in the two dimensions. The delay between scans was 1 s. Typically 96 scans were obtained per free induction decay in all two-dimensional experiments. The NMR data were processed on a Silicon Graphics INDY workstation using Felix 2.3 software (Biosym Technologies, Inc.). Sine-bell squared window functions shifted by 60° followed by 2-Hz exponential multiplication were applied in both dimensions, and the data were zero-filled to 2048 points in t(1) prior to Fourier transformation. Polynomial base-line correction was applied to the F(2) and F(1) dimensions to obtain flat base lines. One-dimensional spectra of samples in 90% H(2)O, 10% D(2)O were also acquired using jump and return water suppression.

Association Constant of Co(III)-Blm A(2) with DNA oligomers

Fluorescence spectra of Co(III)-Blm A(2) were recorded with an SLM AMINO 4800C spectrofluorimeter. Excitation at 300 nm produces an intense emission from the bithiazole moiety at 350 nm, which is quenched upon interaction with DNA. Titration of Form I, the DNA 10-mer, with DNAa was carried out in 20 mM HEPES buffer at pH 7.4 and 25 °C. Plots of the reduction in fluorescence intensity of the drug molecule as a function of added oligomer were analyzed as described previously using the assumption that Co(III)-Blm A(2) binds to equivalent, noninteracting sites on DNA (Chien et al., 1977). From the analysis one can determine K, the association constant, and n, the number of base pairs/binding site, using the following equation,

in which [DNA(o)] represents the total concentration of DNA base pairs.


RESULTS

NMR Features of the Interaction of HO(2)-Co(III)-Blm A(2) (Form I) with d(GGAAGCTTCC)(2) (DNAa)

A combination of two-dimensional NMR techniques has been used to examine the complexes of Form I and II bound to two DNA oligomers incorporating either the GpC or GpT sites of cleavage (Petering et al., 1990). The formation of 1:1 complexes of Form I and DNA was monitored by following the disappearance of DNA imino proton resonances. Both drug-DNA oligomer complexes were in slow exchange on the NMR time scale as illustrated by the observation of distinct resonances for free and bound DNA in the imino region of the spectrum at intermediate drug-to-DNA concentrations for the titration of d(GGAAGCTTCC) with Form I (Fig. 2).


Figure 2: Form I titration. One-dimensional proton NMR titration of 2 mM DNAa with Form I in 20 mM phosphate buffer and 0.1 M NaCl, pH 7.4, and 15 °C.



The binding of Form I lifts the 2-fold symmetry of the DNA and thereby splits the degeneracy of its ^1H NMR spectra. Fig. 3illustrates this for the fingerprint region in the NOESY spectrum of the Form I-DNAa complex, showing a sequential NOE pathway for each DNA strand. The sequential cross-peak between C6-H1` and T7-H6 protons is missing in this region, suggesting that the drug has substantially perturbed this part of the structure. In addition, the sequential cross-peaks between the G15 and T7 imino protons are missing in the H(2)O NOESY spectrum, and these protons are significantly shifted upfield. These results are consistent with the intercalation of the drug between C6-G15 and T7-A14 (Wüthrich, 1986). A list of intermolecular NOEs involving the bithiazole B5 and B5` protons in Table 1confirms this interpretation by showing that these protons interact with several protons in the C6-G15 and T7-A14 base pairs. Together with the upfield shifts in position of the bithiazole B5 and B5` resonances from 8.11 and 7.77 ppm to 7.04 and 6.97 ppm, respectively, these results confirm that the sulfur-containing edge of the bithiazole intercalates between C6-G15 and T7-A14. Similar data have been obtained from an analysis of the Form I-DNAc adduct, leading to the same interpretation (Wu et al., 1994).


Figure 3: Fingerprint region of the ^1H 250-ms NOESY spectrum of the 2 mM 1:1 Form I-DNAa complex in D(2)O, 20 mM phosphate (pH 7.4), and 0.1 M NaCl at 25 °C. a and b trace the sequential NOE pathways for each strand from 5` end to 3`. The dashed circle indicates the missing sequential NOE (C6-H1` to T7-H6).





Form I itself retains almost all of the intramolecular NOEs seen in the absence of DNAa at positions similar to those previously reported (Table 2) (Xu et al., 1994). Thus, in the DNAa adduct, extensive folding exists in the peptide linker region, which generates a close-packed structure involving the metal domain and linker similar to that in free Form I. However, the NOE between B5 and pyrimidinyl (P) methyl hydrogens is missing. In the unbound form this NOE shows that the metal domain is folded over the DNA domain (Xu et al., 1994). Evidently, this interaction is lost upon intercalation of the bithiazole into DNA.



The PCH(3) protons at 2.55 ppm display NOE interactions with an exchangeable base proton at 10.22 ppm. Although highly shifted, this proton is identified as a G5 2-amino proton on the basis of its NOE connections with G5 1-imino and G5 2-amino hydrogens, which are its nearest neighbor protons (Fig. 4A). Thus, the metal domain is closely associated with the guanine base at the specific cleavage site and within a few angstroms of the binding site for the DNA domain. A large shift of imidazole H2 upon binding to DNA from 8.61 to 9.05 ppm also signals that the metal domain interacts with the oligomer. With the bithiazole intercalated between C6-G15 and T7-A14, the metal domain-linker structure intact, and the metal domain in proximity to G5, it appears that Form I retains a compact, folded structure when bound to DNA. Evidently, Form I can bind to a GpC site of cleavage without substantial reorganization of its solution structure, bringing the minor groove cleavage site of the drug into apposition with the DNA and metal domains.


Figure 4: Portions of the jump-return NOESY spectra ((m) = 200 ms) of the 2 mM Form I-DNA (1:1). Spectra were recorded in H(2)O/D(2)O (90/10), 20 mM phosphate (pH 7.4), and 0.1 M NaCl. a, Form I-DNAa, 15 °C; b, Form I-DNAb, 5 °C.



NMR Features of the Interaction of HO(2)-Co(III)-Blm A(2) (Form I) with d(AAACGTTT)(2) (DNAb)

A similar picture is emerging from an examination of the Form I-DNAb adduct. The bithiazole B5 and B5` proton resonances are shifted to a higher field (8.11 and 7.77 ppm to 6.85 and 6.73 ppm, respectively) upon binding of Form I to DNAb. Correspondingly, the T6 and T7 imino protons are upfield shifted, consistent with the intercalation of the B5-B5` edge of the bithiazole between T6-A14 and T7-A13. Intermolecular NOEs between these parts of the two molecules also support the binding of the bithiazole to this region of DNAb (Table 1). In addition, as with the Form I-DNAa complex, the folded structure of the metal domain-linker remains in the DNA complex, but the B5-PCH(3) NOE is missing (Table 2). Instead, the PCH(3) protons make an NOE connection with a G5 2-amino proton (Fig. 4B). Also, the imidazole H2 is highly shifted from 8.69 to 9.14 ppm as in Form I-DNAa. Clearly, Form I binds to both sites, GpC and GpT, with closely related conformations.

NMR Features of the Interaction of Form II, Co(III)-Blm A(2) with d(GGAAGCTTCC)(2)

Form II binds to DNAa in faster exchange than Form I and does not break the 2-fold symmetry of the DNAa NOESY spectrum ( Fig. 5and 6). In data not shown, the fast exchange regions for the adduct existed over the temperature ranges of 5-35 °C. Nevertheless, small perturbations in chemical shifts of protons throughout both the drug and DNA structures indicate that a binding interaction does occur. The maintenance of the degenerate, uninterrupted NOE pathway in Fig. 4shows that Form II does not intercalate into the DNA base structure like Form I. Still, the B5 and B5` protons are shifted upon interaction with DNAa. As with Form I, almost all of the NOEs seen in solution remain unperturbed in the DNA-bound structure (Table 1). There is no NOE connection between PCH(3) and the DNA minor groove as is present in the Form I-DNA structures. Nor have any other intermolecular NOEs been assigned. Furthermore, the highly shifted G5 2-amino proton and H2 proton resonances detected in Form I-DNA adducts are not seen with Form II; instead, they are only modestly perturbed from their solution values (8.58-8.79 and 6.75-6.85 ppm, respectively for H2 and G5 2-amino). Interestingly, the DNA protons in the fingerprint region which are most perturbed by the presence of Form II are those in the GC base pairs at the ends of the 10-mer. Thus, the H6, H5, and H1` protons of C9 are shifted downfield 0.05, 0.05, and 0.06 ppm, respectively, and, correspondingly, those of C10 are shifted, 0.26, 0.29, and 0.09 ppm. Evidently, Form II-DNAa adopts a different conformation than Form I-DNAa-c, while maintaining much, if not all, of the solution conformation of this form of the drug.


Figure 5: Form II titration. One-dimensional proton NMR titration of 2 mM of DNAa with Form II in D(2)O and 20 mM phosphate buffer, pH 7.4, 15 °C.



Association Constant of Co(III)-Blm A(2) with DNAa

The strikingly different properties of binding of Form I and Form II to DNAa prompted the measurement of the association constant of Form II with DNAa. Following a method used previously, Form II was titrated with DNAa and the extent of their interaction determined by the degree of quenching of the bithiazole fluorescence upon binding to the oligomer (Chien et al., 1977). Fig. 7a shows fluorescence emission spectra of Form II in the absence and presence of DNAa. The quenching that was observed is qualitatively similar to that observed when metal-free Blm interacts with DNA (Chien et al., 1977). In Fig. 7, b and c, a summary of the fluorescence titrations is presented, together with the a sample plot of data from one titration based on . Its linearity validates the simple binding model described in . From four titrations the average and standard deviation for the association constant and number of base pairs per binding site were 1.6 ± 0.2 times 10^5M and 10.2 ± 0.6, respectively. The latter figure indicates that a maximum of one drug molecule associated with each 10-mer. Considering the magnitude of this equilibrium constant and the concentrations used in the NMR experiments under similar conditions, virtually all of the Form II added to DNAa during the NMR titration study became bound to the oligomer.


Figure 7: Fluorescence titration of DNAa by Form II. a, emission spectra of Form II and Form II in the presence of a ratio of 1.25:1.00 DNAa to Form II. b, summary of four plots of the decrease in fluorescence emission of Form II as a function of added DNAa. Error bars represent ranges of values. Conditions were as follows: 20 mM HEPES, pH 7.4, and 25 °C. c, double reciprocal plot based on to determine K and n.




DISCUSSION

Detailed structural information is beginning to appear about metallobleomycins bound to DNA. The structure of a Zn-Blm DNA oligomer has been published (Manderville et al., 1995). NMR information about a Form I adduct with 10-mer DNA has also been described (Wu et al., 1994). Although a structure for this complex is not yet in hand, comparative NMR studies offer the opportunity to survey how variations in drug structure and DNA sequences affect adduct formation.

Previous studies have suggested that the DNA and metal domains of ON-Fe(II)-Blm, OC-Fe(II)-Blm, O(2)-Fe(II)- and O(2)-Co(II)-Blm-DNA, and Fe(III)-Blm interact with DNA (Albertini and Garnier-Suillerot, 1984; Antholine and Petering, 1979; Antholine et al., 1981; Chikira et al., 1989; Fulmer and Petering, 1994). Features of the Form I-oligonucleotide structure described above confirm the intimate interaction of both drug domains with DNA. Indeed, in each of the three Form I-DNA structures that have been examined, it is an element of the metal domain that associates with the G5 part of the preferred GpC or GpT site of cleavage. The DNA domain also intercalates between several stacks of base pairs, involving C6-T7, T6-T7, and C6-C7, so the site of the primary interaction of bithiazole is not unique but does include a member of the cleavage site on the 3` side (Wu et al., 1994).

Knowledge of Form I-DNA conformation is derived from several types of NMR information. The intramolecular NOEs in Table 2show that Form I retains its solution conformation for the linker and metal domain when bound to DNAa and DNAb. The intermolecular NOEs as well as the upfield chemical shifts of the bithiazole protons and protons of the base pairs involving bases at positions 6 and 7 indicate that the bithiazole intercalates into the base pair structure of all three Form I-DNA structures ( Table 1and Wu et al.(1994)). The finding that the PCH(3) group in the metal domain is in close proximity to G5 in each structure supports the close association of the metal domain with DNA (Fig. 4). In the Form I complex with d(CCAGGCCTGG)(2), the assignment of this NOE to an interaction between a G4 or G5 2-amino proton and PCH(3) protons could not be distinguished (Wu et al., 1994). The presence of the same NOE in all three Form I-DNA complexes now supports the identification of this NOE as a G5 2-amino proton-PCH(3) proton interaction in each complex.

A synthesis of these structural determinants shows that Form I exists in each DNA complex as a structure in which the metal domain and linker are folded similarly to those found in the absence of DNA. Using the unbound structure as a model, the metal domain is in close association with G5, the peroxide coordinated to Co occupies a position between G5 and T6 or C6, and the bithiazole interacts between the base pairs at positions 6 and 7. Evidently, the metal domain as well as the DNA domain interacts with DNA as previously hypothesized and participates in site selectivity at the guanine residue of the GpC and GpT pairs (Xu et al., 1994). This conclusion is buttressed by the finding that the chemical shift of the histidine H2 proton, part of the metal domain, is markedly perturbed in all of the DNA adducts. A role for the metal domain in site selectivity has been previously hypothesized (Carter et al., 1990; Kane and Hecht, 1994).

The Form I-DNA conformation is not unique among metallobleomycins bound to DNA; a different one has been reported for Zn-Blm-DNA (Manderville et al., 1994, 1995). On the basis of the present experiments, the binding mode of Form II to DNAa is also distinct from that of Form I. Furthermore, Form II associates with DNA in a conformation that includes extensive folding in the linker region that has not been observed for Zn-Blm either in solution or when bound to DNA (Akkerman et al., 1988, Manderville et al., 1994, 1995).

Fluorescence titration experiments have shown that Form II binds to DNAa with an association constant comparable with ones measured for bleomycin and similar to or 10-fold larger than values for Cu-Blm under conditions of low ionic strength (Chien et al., 1977; Povirk et al., 1981; Roy et al., 1981). At 1.7 times 10^5M, it is sufficiently large to insure that virtually all of the detectable drug binds to DNA with the concentrations and ionic strength used in the NMR experiments (Fig. 7). It is also large enough to question whether the fast exchange behavior of Form II in the NMR experiments seen in Fig. 5results from rapid exchange of Form II either bound to a particular site on the 10-mer or free in solution.

An attractive alternative is that Form II slides along the 10-mer such that drug exchanges rapidly between sites on the oligomer while bound to it. Other examples of sliding by ligands binding to DNA oligomers have been hypothesized based on NMR studies in which association between ligand and DNA does not break the symmetry of the DNA NMR spectrum (Leupin et al., 1986; Pelton and Wemmer, 1990). In the present study, the finding that DNA proton chemical shifts were most perturbed in the GC base pairs at the ends of the molecule may also be consistent with this interpretation (data not shown). The lack of any assignable intermolecular NOEs suggests that sliding is very fast, thereby reducing the intensity of NOEs related to any particular drug-site interaction. Because the fast exchange binding of Form II to DNA is qualitatively similar to that of Zn-Blm, the latter complex may also be able to slide along the DNA molecule (Manderville et al., 1994, 1995).

The possibility that Form II can slide quickly along DNA stands in contrast to the apparent static binding of O(2)-Co(II)-Blm to DNA (Xu et al., 1992b). The latter permits such molecules in close proximity along the DNA structure to exist for extended periods without undergoing bimolecular oxidation reduction to produce Form I and Form II (Xu et al., 1992a). It is hypothesized that O(2)-Co(II)-Blm is folded, possibly like Form I, when bound to DNA (Chikira et al., 1989). The hypothesis that Form II may move along the DNA structure while bound to it suggests that sliding by extended metallobleomycin structures might play a role in the mechanism by which sites are selected for DNA cleavage by the drug.

A major question arises from these findings: why does Form II not bind to DNA like Form I? Each contains the same DNA domain structure with its bithiazole and positively charged tail, which provides electrostatic stabilization for the drug-DNA adduct. The net charge on the metal domain of Form I is 1+; the charge of the metal domain of Form II is either 1 or 2+, depending on the charge on the ligand occupying its sixth coordination position, probably either water or hydroxide. Therefore, the positively charged metal domains of both forms should interact favorably with the negative charge of the DNA polymer and not be an obvious source of discrimination between them. A possible answer to this question relates to the fact that Forms I and II may adopt different structures in solution; the first has the bithiazole folded over the metal domain, and the second has the bithiazole tail region extended from the folded metal domain-linker region (Xu et al., 1994). Apparently, these conformations substantially remain in their DNA adducts. According to molecular dynamics calculations on Form I and Form II, the ligand structure in each wraps oppositely about Co(III) to yield two chiral metal domains (Xu et al., 1994). One result of such differential folding is that the two metal domains cannot identically interact with DNA (Xu et al., 1994). This conclusion is consistent with the differential behavior of the H2 and G5 2-amino protons in the two DNA adducts and the lack of an NOE connection between the PCH(3) protons and a G5 2-amino proton in the Form II-DNA complex. A related outcome is that the position of the bulky disaccharide is different in the two conformations. A third consequence of the different chiralities is that only in Form I can the bithiazole fold back upon the metal domain in Form I, placing the peroxide between them. In contrast, in Form II it is only possible to fold the DNA domain over the metal domain such that the sixth ligand position is on an outer face of the metal domain, not between them, making it impossible for those two structures to bind similarly to DNA. Therefore, it is suggested that intercalation by the bithiazole requires a specific metal domain configuration, which permits a particular folded conformational relationship to exist between the metal and DNA domains. The reason for this requirement needs further study.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant 22184. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Chemistry, University of Wisconsin-Milwaukee, P.O. Box 413, Milwaukee, WI 53201.

(^1)
The abbreviations used are: Blm, bleomycin; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy.


ACKNOWLEDGEMENTS

We appreciate the use of the spectrofluorimeter in the laboratory of Dr. Sally Twining.


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