Different Conformations and Site Selectivity of HO2−-Co(III)-Bleomycin A2 and Co(III)-Bleomycin A2 Bound to DNA Oligomers

Qunkai Mao,Patricia Fulmer,Wenbao Li,Eugene F Derose,David H Petering

doi:10.1074/jbc.271.11.6185

Qunkai Mao, Patricia Fulmer + Show 3 more

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https://doi.org/10.1074/jbc.271.11.6185

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Abstract

Conformational properties of HO2(-)-Co(III)-bleomycin A2 (Form I) and Co(III)-bleomycin (Form II) bound to DNA oligomers offering either principal cleavage site for the drug, d(GGAAGCTTCC)2 or d(AAACGTIT)2, 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 degrees 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)2 in 20 mM HEPES buffer at pH 7.4 and 25 degrees C is 1.7 x 10(5) M(-1), and 1.0 mol of Form II bind per mol of 10-mer.

Highlights

The redox chemistry of Fe-Blm in solution or bound to DNA can be modeled closely by Co-Blm (Xu et al, 1992a, 1992b)
NMR data on the interaction of Form I with DNA 10-mer, d(CCAGGCCTGG)2 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)
NMR Features of the Interaction of HO2Ϫ-Co(III)-Blm A2 (Form I) with d(GGAAGCTTCC)2 (DNAa)—A combination of two-dimensional NMR techniques has been used to examine the complexes of Form I and II bound to two DNA oligomers incorporating either the GpC or GpT sites of cleavage (Petering et al, 1990)

Summary

The abbreviations used are

Bleomycin; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy. can be modeled closely by Co-Blm (Xu et al, 1992a, 1992b). 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 Form I the bithiazole moiety is folded back over the metal domain These structures bind tightly to DNA, offering the opportunity to examine them complexed with DNA as models for the related Fe-Blms (Xu et al, 1992b). 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). It 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

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DISCUSSION