STUDIES OF CO-CENTER-DOT-BLEOMYCIN A2 GREEN - ITS DETAILED STRUCTURALCHARACTERIZATION BY NMR AND MOLECULAR MODELING AND ITS SEQUENCE-SPECIFIC INTERACTION WITH DNA OLIGONUCLEOTIDES

The structure of homogeneous Co Bleomycin (CoBLM) A2 green (the hydrop eroxide form of CoBLM) has been determined using 2D NMR methods and mo lecular dynamics calculations. Previous studies of Xu et al. (Xu, R. X .; Nettesheim, D.; Otvos, J. D.; Petering, D. H. Biochemistry 1994, 33 , 907-916) reported several possible structures for CoBLM A2 green com patible with their NMR data acquired on a mixture of CoBLM A2 green an d A2 brown forms. The availability of the pure CoBLM A2 green, which i s stable for months at neutral pH, has allowed the complete assignment s of the H-1 and C-13 chemical shifts, observation of 55 intramolecula r NOEs, and determination of 15 coupling constants allowing the defini tion of dihedral angles. These results are a prerequisite to determini ng its structure with duplex DNA of a defined sequence (Wu, W.; Vander wall, D. E.; Turner, C. J.; Kozarich, J. W.; Stubbe, J. J. Am. Chem. S ec. 1996, 118, 1281-1294). Two screw sense isomers each containing two possible axial ligands (the primary amine of the beta-aminoalanine an d the carbamoyl nitrogen of the mannose) were considered as viable can didates for the structure of CoBLM A2 green. Using the NMR constraints and molecular dynamics calculations, the structures of all four isome rs were generated. One set of screw sense isomers can be readily elimi nated from considerations based on violations of NOE and dihedral angl e constraints. The other screw sense isomer containing either one or t he other of the postulated axial ligands has been examined in some det ail. The structure containing the primary amine of beta-aminoalanine a s the axial ligand is favored on the basis of coupling constants and N OE arguments, potential energy considerations, model studies, and stud ies with analogs of BLM. The favored structure is compact with the bit hiazole moiety folded back underneath the equatorial plane of the meta l binding domain, on the same face as the hydroperoxide ligand. The ge ometry of the peptide Linker is very well defined by the observed coup ling constants in the valeryl and threonine moieties of the linker. Co BLM A2 green has been studied with two self-complementary oligonucleot ides, d(CCAGGCCTGG) and d(CCAGTACTGG). Both of these oligomers possess a single, UV light-mediated cleavage site (C and T, respectively). In addition, fluorescent quenching studies have allowed the determinatio n of the first sequence-specific dissociation constants of 1.7 x 10(-7 ) and 1.5 x 10(-7) M, respectively. Titration of CoBLM A2 green with e ach of these oligomers reveals a 1:1 complex in slow exchange on the N MR time scale. The upfield shifts of the bithiazole protons in both of these complexes are indicative of a partial intercalative mode of bin ding. The stage is now set for the determination of the structure of t he CoBLM A2 green bound sequence specifically to DNA.