Solution Structure of Co·Bleomycin A2 Green Complexed with d(CCAGGCCTGG)

The solution structure of Co·Bleomycin (CoBLM) A2 green
(the hydroperoxide form of CoBLM) complexed
with the self-complementary oligonucleotide d(CCAGGCCTGG)
with a cleavage site at C6 has been determined
by
2D NMR spectroscopic methods and molecular dynamics calculations.
Intermolecular NOEs (60 between CoBLM
A2 green and DNA) and intramolecular NOEs (61 within CoBLM A2 green)
have defined the position and orientation
of CoBLM A2 green with respect to its single binding site in the
duplex. CoBLM A2 green is a stable analog of
the activated BLM, the Fe3+ hydroperoxide (Sam, J. W.;
Tang, X.-J.; Peisach, J. J. Am. Chem.
Soc. 1994, 116,
5250−5256). These studies have provided the first structural
insight into the mode of binding of the bithiazole tail
of CoBLM A2 green to DNA, the basis for specificity of its cleavage at
pyrimidines (Py) in d(G-Py) sequences, and
the orientation of its terminal oxygen of the hydroperoxide relative to
the 4‘ carbon hydrogen bond being cleaved in
the DNA. The bithiazole tail inserts 3‘ to the C6 cleavage site
from the minor groove. The terminal thiazolium ring
is completely stacked between the bases of G14 and G15, while the
penultimate thiazolium ring is only partially
stacked between the bases of C6 and C7. The bithiazole tail thus
binds via a partial intercalation mode and the
DNA is unwound by 13° over the
(G5·C16)∼(C6·G15)∼(C7·G14)∼(T8·A13)
steps. No specific interactions between
the bithiazole tail and the DNA have been identified, and thus, this
interaction does not define the BLM's cleavage
specificity but its binding affinity. The metal binding domain and
the peptide linker region of CoBLM A2 green
bind within the minor groove of the duplex and define the basis for its
specificity of DNA cleavage. The 4-amino
group and the N3 of the pyrimidine ring of CoBLM A2 green form specific
hydrogen bonds with the N3 and the
2-amino group, respectively, of the G5 in the duplex and provide an
unusual example of a minor groove base triple-like interaction. A basis for the preference for G over A, 5‘ to
the Py cleavage site, is thus established. The metal
binding domain and the valeryl moiety in the linker have a conformation
strikingly similar to that defined in the free
CoBLM A2 green (Wu, W.; Vanderwall, D. E.; Lui, S. M.; Tang, X.-J.;
Turner, C. J.; Kozarich, J. W.; Stubbe, J.
J. Am. Chem. Soc.
1996, 118, 1268−1280). The most remarkable
feature of this structure is the observation of the
proton associated with the hydroperoxide of CoBLM A2 green and its
observed intermolecular NOEs to the minor
groove protons of C6 and C7 of the duplex. Thus this structure
provides a rare snapshot of an analog of a reactive
intermediate poised to initiate the hydrogen atom abstraction event.
The molecular modeling reveals that the distal
oxygen of the hydroperoxide is 2.5 Å from the 4‘-hydrogen of C6.
A number of additional intramolecular hydrogen
bonds between the hydroperoxide ligand and the peptide linker region
are also proposed, which appear to play a key
role in positioning the reactive intermediate near the hydrogen atom
being abstracted. This structural model makes
a number of predictions that can be tested experimentally given the
recent modular synthesis of BLM (Boger, D. L.;
Teramoto, S.; Honda, T.; Zhou, J. J. Am.
Chem. Soc. 1995, 117,
7338−7343).