(Received for publication, September 21, 1995; and in revised form, December 13, 1995)
From the
Conformational properties of
HO-Co(III)-bleomycin A
(Form
I) and Co(III)-bleomycin (Form II) bound to DNA oligomers offering
either principal cleavage site for the drug, d(GGAAGCTTCC)
or d(AAACGTTT)
, 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)
in 20 mM HEPES buffer at pH 7.4 and 25 °C is 1.7
10
M
, and 1.0 mol of
Form II bind per mol of 10-mer.
Bleomycin (Blm) ()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
and reductants to generate
HO
-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-Co(III)-Blm (Form I) and Co(III)-Blm
(Form II) or the corresponding iron species. However, in contrast to
HO
-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) 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.
in which [DNA] represents the total
concentration of DNA base pairs.
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 H 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
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 H
250-ms NOESY spectrum of the 2 mM 1:1 Form I-DNAa complex in
D
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 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
( = 200 ms) of the 2 mM Form I-DNA
(1:1). Spectra were recorded in H
O/D
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.
Figure 5:
Form
II titration. One-dimensional proton NMR titration of 2 mM of
DNAa with Form II in DO and 20 mM phosphate
buffer, pH 7.4, 15 °C.
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.
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-Fe(II)- and
O
-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 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)
, the assignment of this NOE to an interaction
between a G4 or G5 2-amino proton and PCH
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
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
10
M
, 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-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
-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 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.
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