Solution structure of Co center dot 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. Ant. Chem. Sec. 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 degrees over the (G5 . C16)similar to(C6 . G15)similar to(C7 . G14)similar to(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 LF-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. Sec. 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 Angstrom 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. Sec. 1995, 117, 7335-7343).