The antitumor antibiotic bleomycin (BLM) binds to and degrades the self-complementary octanucleotide d(CGCTAGCG)2 in a sequence-selective fashion. To model the binding interaction, 1:1 complexes of Zn(II)BLM A2 and Zn(II)·BLM A5 with the DNA oligonucleotide have been examined using two-dimensional NMR experiments and restrained molecular dynamics calculations. Intercalation is indicated by the broadening and upfield shifting of the BLM aromatic bithiazole protons and DNA base-paired imino protons. However, the data do not support a classical mode of intercalation, as the sequential intrastrand NOE connectivities of d(CGCTAGCG)2 are not disrupted upon Zn·BLM binding. The orientation of the drug molecule in the helix is based on the finding of eight intermolecular BLM–DNA NOEs in the Zn·BLM A5–d(CGCTAGCG)2 complex. The bithiazole B-ring proton (Bit 5) and spermidine H3 (Sp 3) atoms are positioned within 5 Å of adenosine5 H2 in the minor groove, while the bithiazole A-ring proton (Bit 5’) shows major groove contacts to protons of cytidine3 and thymidine4. Protons of the β-hydroxyhistidine and methyl valerate residues show minor groove contacts (H4′ and H5′,5″) to cytidine7. Using the NMR-derived NOE distance and dihedral bond angle restraints to guide the molecular dynamics calculations, a binding model for the interaction of Zn•BLM with the octanucleotide was derived. To satisfy the major and minor groove BLM-DNA NOE contacts, this model positions the bithiazole ring system in a “cis” orientation (H atoms on same side) with the H atoms directed into the helix. Such an orientation favors partial stacking interactions between the DNA bases and the bithiazole rings and permits interaction of the cationic spermidine tail and metal binding domain with the minor groove of the helix. The upfield shifts observed for the bithiazole aromatic protons are in agreement with the effects predicted for protons oriented as in the model. The BLM molecule adopts a folded conformation that favors H bond formation between the exocyclic NH2 group of guanosine6 and the hydroxyl group of the methyl valerate residue. This binding mode involves DNA unwinding, which widens the minor groove, as well as a bend in the helix that is induced at the bithiazole binding site. The DNA unwinding angle of 13° calculated for this model is in good agreement with the experimentally determined value of 12°. The validity of this DNA binding model and possible implications for sequence-selective cleavage by BLM are discussed. Also discussed is the effect of DNA sequence on the nature of BLM–DNA interaction.
ASJC Scopus subject areas
- Colloid and Surface Chemistry