Crystal and solution structures of 7-amino-actinomycin D complexes with d(TTAGBrUT), d(TTAGTT) and d(TTTAGTTT)

Synopsis: In the crystal, actinomycin D intercalates between G-T wobble pairs of DNA involving the sequence d(TTAGTT); in solution, the dominant binding is between guanine and actinomycin. Abstract: The formation of the complex of 7-amino-actinomycin D with potentially single-stranded DNA has been studied by X-ray crystallography in the solid state, by NMR in solution, and by molecular modeling. The crystal structures of the complex with 5'-TTAG[Br5U]T-3' provide interesting examples of MAD phasing, in which the dispersive component of the MAD signal was almost certainly enhanced by radiation damage. The trigonal and orthorhombic crystal modifications both contain antibiotic molecules and DNA strands in the form of a 2:4 complex; in the orthorhombic form there is one such complex in the asymmetric unit, in the trigonal structure there are four. In both structures the phenoxazone ring of the first drug intercalates between a BrU-G (analogous to T-G) wobble pair and a G-T pair where the T is part of symmetry related molecule. The chromophore of the second actinomycin intercalates between BrU-G and G-BrU wobble pairs of the partially paired third and fourth strands. The base stacking also involves (A*T)*T triplets and Watson-Crick A-T pairs and leads to similar complex three-dimensional networks in both structures with looping-out of unpaired bases. Although the available NOE-constraints of a solution containing the antibiotic and d(TTTAGTTT) strands in the ratio 1:1 are insufficient for determining the structure of the complex from the NMR data alone, they are consistent with the intercalation geometry observed in the crystal structure. Molecular dynamics (MD) trajectories starting from the 1:2 complexes observed in the crystal showed that although the thymines flanking the d(AGT) core are rather flexible and the G-T pairing is not permanently preserved, both strands remain bound to the actinomycin by strong interactions between it and the guanines between which it is sandwiched. Similar strong binding (hemi-intercalation) of the actinomycin to a single guanine was observed in the MD trajectories of a 1:1 complex. The dominant interaction is between the antibiotic and guanine, but the complexes are stabilized further by promiscuous base-pairing