Solution Structure of Co(III)-Bleomycin-OOH Bound to a Phosphoglycolate Lesion Containing Oligonucleotide:  Implications for Bleomycin-Induced Double-Strand DNA Cleavage

Abstract

Bleomycin (BLM) is an antitumor antibiotic that is used clinically. Its major cause of cytotoxicity is thought to be related to BLM's ability to cause double-strand (ds) DNA cleavage. A single molecule of BLM appears to cleave both strands of DNA in the presence of its required cofactors Fe(2+) and oxygen without dissociating from the helix. A mechanism for this process has been proposed based on a model structure of the hydroperoxide of Co(III)-BLM (CoBLM) bound sequence-specifically to an intact duplex containing a GTAC site, a hot spot for ds cleavage [Vanderwall, D. E., Lui, S. M., Wu, W., Turner, C. J., Kozarich, J. W., and Stubbe, J. (1997) Chem. Biol. 4, 373-387]. In this paper, we present a structural model for the second cleavage event. Two-dimensional NMR spectroscopy and molecular modeling were carried out to study CoBLM bound to d(CCAAAGXACTGGG).d(CCCAGTACTTTGG), where X represents a 3'-phosphoglycolate lesion next to a 5'-phosphate. Assignments of 729 NOEs, including 51 between the drug and the DNA and 126 within the BLM molecule, have been made. These NOEs in addition to 96 dihedral angle constraints have been used to obtain a well-defined structural model for this complex. The model reveals that the bithiazole tail is partially intercalated between the T19 and the A20 of the duplex and that the metal binding domain is poised for abstraction of the T19 H4' in the minor groove. The modeling further reveals that the predominant conformation of the bithiazole protons is trans. Two cis conformations of these protons are also observed, and ROESY experiments provide evidence for interconversion of all of these forms. The relationship of these observations to the model for ds cleavage is presented.

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

“…Together, the interactions of the metal-binding domain and disaccharide with the minor groove bury a surface area of 818 Å 2 , whereas the interaction between the ␣-D-mannose moiety, and the metal-binding domain buries 295 Å 2 . Based on coordination constraints and comparison with NMR-derived structural models (23,24), a model of the hydroperoxide ligand places the distal oxygen 2.5 Å from the C4Ј atom of T 6 and the coordinating oxygen 4.7 Å from the same C4Ј atom (Fig. 5); this finding is entirely consistent with the observations published in refs.…”

“…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).…”

“…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.…”

Definition of the intermediates and mechanism of the anticancer drug bleomycin using nuclear resonance vibrational spectroscopy and related methods

“…The initial Cartesian coordinates were taken from NMR structures [PDB 1GJ2 (43) and 1MXK (27)] of ðBLMÞCo III ─OOH, and the Co was replaced by Fe and the nature of the axial ligand was varied as described below. The DNA binding domain and carbohydrate domain (Fig.…”