INTERACTION OF ZN(II)CENTER-DOT-BLEOMYCIN WITH D(CGCTAGCG)(2) - A BINDING MODEL-BASED ON NMR EXPERIMENTS AND RESTRAINED MOLECULAR-DYNAMICS CALCULATIONS

The antitumor antibiotic bleomycin (BLM) binds to and degrades the sel f-complementary octanucleotide d(CGCTAGCG)(2) in a sequence-selective fashion. To model the binding interaction, 1:1 complexes of Zn(II). BL M A(2) and Zn(II). BLM A(5) with the DNA oligonucleotide have been exa mined using two-dimensional MMR experiments and restrained molecular d ynamics 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 mod e of intercalation, as the sequential intrastrand NOE connectivities o f d(CGCTAGCG)(2) are not disrupted upon Zn . BLM binding. The orientat ion of the drug molecule in the helix is based on the finding of eight intermolecular BLM-DNA NOEs in the Zn . BLM A(5)-d(CGCTAGCG)(2) compl ex. The bithiazole B-ring proton (Bit 5) and spermidine H3 (Sp 3) atom s are positioned within 5 Angstrom of adenosine(5) H2 in the minor gro ove, while the bithiazole A-ring proton (Bit 5') shows major groove co ntacts to protons of cytidine(3) and thymidine(4). Protons of the beta -hydroxyhistidine and methyl valerate residues show minor groove conta cts (H4' and H5',5'') to cytidine(7). Using the NMR-derived NOE distan ce 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 interacti ons between the DNA bases and the bithiazole rings and permits interac tion of the cationic spermidine tail and metal binding domain with the minor groove of the helix. The upfield shifts observed for the bithia zole aromatic protons are in agreement with the effects predicted for protons oriented as in the model. The BLM molecule adopts a folded con formation that favors H bond formation between the exocyclic NH2 group of guanosine(6) and the hydroxyl group of the methyl valerate residue . This binding mode involves DNA unwinding, which widens the minor gro ove, as well as a bend in the helix that is induced at the bithiazole binding site. The DNA unwinding angle of 13 degrees calculated for thi s model is in good agreement with the experimentally determined value of 12 degrees. The validity of this DNA binding model and possible imp lications for sequence-selective cleavage by BLM are discussed. Also d iscussed is the effect of DNA sequence on the nature of BLM-DNA intera ction.