The structure of homogeneous Co·Bleomycin (CoBLM) A2 green
(the hydroperoxide form of CoBLM)
has been determined using 2D NMR methods and molecular 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 compatible with their NMR data acquired
on a mixture of CoBLM A2 green and
A2 brown forms. The availability of the pure CoBLM A2 green, which
is stable for months at neutral pH, has
allowed the complete assignments of the 1H and
13C chemical shifts, observation of 55 intramolecular NOEs,
and
determination of 15 coupling constants allowing the definition of
dihedral angles. These results are a prerequisite
to determining its structure with duplex DNA of a defined sequence (Wu,
W.; Vanderwall, D. E.; Turner, C. J.;
Kozarich, J. W.; Stubbe, J. J. Am. Chem. Soc.
1996, 118, 1281−1294). Two screw sense
isomers each containing
two possible axial ligands (the primary amine of the β-aminoalanine
and the carbamoyl nitrogen of the mannose)
were considered as viable candidates for the structure of CoBLM A2
green. Using the NMR constraints and molecular
dynamics calculations, the structures of all four isomers were
generated. One set of screw sense isomers can be
readily eliminated from considerations based on violations of NOE and
dihedral angle constraints. The other screw
sense isomer containing either one or the other of the postulated axial
ligands has been examined in some detail.
The structure containing the primary amine of β-aminoalanine as
the axial ligand is favored on the basis of coupling
constants and NOE arguments, potential energy considerations, model
studies, and studies with analogs of BLM.
The favored structure is compact with the bithiazole moiety folded
back underneath the equatorial plane of the metal
binding domain, on the same face as the hydroperoxide ligand. The
geometry of the peptide linker is very well
defined by the observed coupling constants in the valeryl and threonine
moieties of the linker. CoBLM A2 green
has been studied with two self-complementary oligonucleotides,
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 determination of the first
sequence-specific dissociation constants of 1.7 ×
10-7
and 1.5 × 10-7 M, respectively.
Titration of CoBLM A2 green with each of these oligomers reveals a
1:1 complex
in slow exchange on the NMR time scale. The upfield shifts of the
bithiazole protons in both of these complexes
are indicative of a partial intercalative mode of binding. The
stage is now set for the determination of the structure
of the CoBLM A2 green bound sequence specifically to DNA.