Interaction of the anthracycline antibiotic nogalamycin with the hexamer duplex d(5'-GACGTC)2. An NMR and molecular modelling study.

The interaction of the anthracycline antibiotic nogalamycin with the single 5'-CpG binding site at the centre of the hexamer duplex d(GACGTC)2 has been investigated in solution by NMR spectroscopy. 41 NOE constrains between antibiotic and nucleotide protons provide input parameters for the determination of a family of conformers computed using energy minimisation and restrained molecular dynamics. Nogalamycin adopts a 'threaded' binding orientation in which the drug chromophore passes through the DNA helix positioning the nogalose sugar in the minor groove and the positively charged bicycloamino sugar in the major groove. The complex is stabilised by hydrogen bonding, electrostatic and van der Waals interactions. In the major groove hydrogen bonding interactions are identified between the 2'-OH and 4'-OH groups, on the rigidly oriented bicyclo sugar, and the guanine N7 and cytosine 4-NH2 of one G.C base pair and appear to account for the requirement for a G.C base pair at the intercalation site. In the minor groove we identify a hydrogen bond between the carbonyl oxygen of the methyl ester group on ring A and the 2-NH2 group of guanine at the intercalation site. The nogalose and ring A form a continuous domain that interacts through van der Waals contacts with the floor and walls of the minor groove over a span of four basepairs, d(GACGTC)2. The NOE data together with energy-minimised structures reveal distortions of the DNA conformation involving a total helix unwinding of about 16 degrees and buckling of the G.C base pairs by about 22 degrees and -9 degrees at the intercalation site. The base pairs effectively wrap around the drug chromophore optimising van der Waals contacts between the two and minimising cavities in the structure. The solution data also reveal that the bound antibiotic substantially slows the rate of exchange of the imino protons of not only the G.C base pairs forming the intercalation site but also that of an A.T base pair which is occluded by the sugar moieties lying in each groove. The data present an opportunity for comparison with two crystal structures of nogalamycin-DNA complexes in which ligand molecules are bound at terminal 5'-CpG sites.