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

Abstract

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 Fe 3+ 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 triplelike 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. ). 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 abstr...

Crystal structure of DNA-bound Co(III)·bleomycin B 2 : Insights on intercalation and minor groove binding

“…The screw sense and Co(III)-coordinating atoms observed in our structure are the same as those proposed in the NMR structures of HOO-Co(III)⅐bleomycin bound to DNA (21,23,24), although the structures clearly differ in detail (Figs. S1-S3).…”

“…S1-S3). More recently, the primary amine of ␤-aminoalanine has been identified definitively as an axial ligand to Co(III) (34) consistent with earlier models (21,23,24).…”

“…In NMR derived models, neither the ␣-D-mannose nor the gulopyranose make direct contacts with the DNA (21,23,24). In our structure, no interactions were observed between the gulopyanose moiety and the DNA, but the ␣-D-mannose carbamoyl NH 2 hydrogen-bonds to the O 2 of T 13 in the minor groove.…”

“…In 2D NMR-based models, hydrogen-bonding interactions between the Thr-NH and methylvalerate-NH of the linker and the penultimate oxygen of the metal-bound peroxide have been proposed to play an important role in preorganizing the structure of HOO-Co(III)⅐bleomycin (21,23,24). However, these same linker amides in our structure point away from the penultimate oxygen of the hydroperoxide and are therefore not positioned to hydrogen bond to the hydroperoxide ligand (Fig.…”

“…1) (15) constitutes a stable structural analogue of O 2 activated Fe(II)⅐bleomycin, HOO-Fe(III)⅐bleomycin, that interacts with the same DNA site-selectivity (16)(17)(18)(19)(20) but requires photoactivation to cleave DNA. From 2D NMR derived models reported for DNA-bound HOO-Co(III)⅐bleomycin, the bithiazole is intercalated and the pyrimidinyl ring of the metal-binding domain interacts with the guanine of the 5Ј-GC site via the minor groove (21)(22)(23)(24)(25). Although these studies have provided key information, the impact of drug binding on the substrate DNA structure has not been fully addressed.…”