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

Abstract

Bleomycins constitute a widely studied class of complex DNA cleaving natural products that are used to treat various cancers. Since their first isolation, the bleomycins have provided a paradigm for the development and discovery of additional DNA-cleaving chemotherapeutic agents. The bleomycins consist of a disaccharide-modified metal-binding domain connected to a bithiazole/Cterminal tail via a methylvalerate-Thr linker and induce DNA damage after oxygen activation through site-selective cleavage of duplex DNA at 5-GT/C sites. Here, we present crystal structures of two different 5-GT containing oligonucleotides in both the presence and absence of bound Co(III)⅐bleomycin B 2. Several findings from our studies impact the current view of bleomycin binding to DNA. First, we report that the bithiazole intercalates in two distinct modes and can do so independently of well ordered minor groove binding of the metal binding/disaccharide domains. Second, the Co(III)-coordinating equatorial ligands in our structure include the imidazole, histidine amide, pyrimidine N1, and the secondary amine of the ␤ aminoalanine, whereas the primary amine acts as an axial ligand. Third, minor groove binding of Co(III)⅐bleomycin involves direct hydrogen bonding interactions of the metal binding domain and disaccharide with the DNA. Finally, modeling of a hydroperoxide ligand coordinated to Co(III) suggests that it is ideally positioned for initiation of C4-H abstraction.antitumor ͉ DNA-binding ͉ HOO-Co(III) bleomycin T he bleomycins are a family of glycopeptide-derived antitumor natural products first isolated from Streptomyces verticillus by Umezawa (1). Bleomycins are used to treat lymphomas (2, 3) and testicular cancers (4), most often in combination therapies. The clinical efficacy of the bleomycins is thought to derive from their ability to mediate single-and double-strand DNA cleavage (5, 6) resulting from Fe 2ϩ and O 2 -dependent (7) C4Ј-H abstraction from pyrimidine nucleotides (8) contained within 5Ј-GT/C dinucleotide sites (9). Of the DNA lesions formed, double-strand DNA cleavage is thought to be most relevant to the cytotoxicity of bleomycin. Although DNA is the generally accepted locus of drug activity, O 2 -activated Fe(II)⅐bleomycin also mediates the selective cleavage of RNA (10), and Cu(I)⅐bleomycin is capable of cleaving DNA (11); however, the in vivo relevance of this alternative nucleic acid target and metal ion remain to be fully determined.The metallobleomycins have served as an important paradigm for nucleic acid-targeted drug design (12,13); and yet, despite extensive study over the past 40 years, fundamental aspects of their activity have yet to be fully defined including the role of particular DNA-binding modes on drug activity and the mechanism of double-strand DNA cleavage (14). Facilitating structural studies, HOO-Co(III)⅐bleomycin ''green'' (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 (16-20)...

In Vivo Manipulation of the Bleomycin Biosynthetic Gene Cluster in Streptomyces verticillus ATCC15003 Revealing New Insights into Its Biosynthetic Pathway

“…This observation may originate from the carbamoyl group directly contributing to the coordination of the metal ion for DNA cleavage or from being involved in DNA binding. Although there has been speculation about the former hypothesis (41), the latter is supported by the recently published crystal structure of DNA-bound Co(III)-BLM B2; the carbamoyl NH 2 forms a hydrogen bond with the minor grove, whereas the function of the disaccharide seems to be to correctly position and stabilize the entire complex (42). In addition to the likely reduced DNA affinity of 3-MOP-decarbamoyl-BLM caused by the lack of the carbamoyl group, the different terminal amines of 3-MOP-decarbamoyl-BLM, BLM A2, and B2 may also contribute to the variation in DNA cleavage activity.…”

Possible structural role of the disaccharide unit in Fe-bleomycin before and after oxygen activation

“…This outcome is not surprising if considered in light of the crystal structure of HOOCo(III)BLM bound to a DNA fragment. 28,32 In this crystal structure the metal-binding domain and disaccharide moieties partially stack against each other and display base-specific hydrogen bonding to each respective wall of the DNA minor groove. The M carbamoyl amide and its C-OH form hydrogen bonds with the DNA bases that are very important for the stabilization of the HOO-Co(III)BLM-DNA complex.…”

“…25 The results of these studies have led to the general agreement that the secondary amine of b-aminoalanine (A) segment, the pyrimidinylpropionamide (P) and imidazole rings, and the amide nitrogen in b-hydroxyhistidine (H) are the equatorial ligands to the metal centers in most of the metal-BLM adducts studied to date. 25 Crystal structures of Cu(II)P3-A, 26 Cu(II)BLM complexed with the BLM-binding protein BLMA, 27 and HOO-Co(III)BLM bound to DNA fragments 28 have pointed the primary amine in A as the endogenous axial ligand to the corresponding metal centers. In the case of the ferrous complex of BLM, no crystal structure exists and there is some controversy concerning the ligation of BLM to Fe(II).…”

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

“…BLM provides five nitrogen-based ligands ( Fig. 1 A and B) for coordination to the metal ion based on a crystal structure of peroxy Co III BLM (7). The four equatorial ligands are an imidazole nitrogen, a deprotonated amide, a pyrimidine nitrogen, and the secondary amine of the β-aminoalanine.…”