The antitumor drug actinomycin D intercalates into DNA and strongly inhibits RNA transcription at very low concentration. Two independent structures of d(GAAGCTTC)(2)-actinomycin D complexes have been determined: by the X-ray single crystal diffraction method (cell dimension a = 69.69, b = 61.41, and c = 54.25 Angstrom; space group F222; R-factor = 0.202 at 3.0-Angstrom resolution). The complexes have an exact 2-fold symmetry in the structure. The two independent complexes are basically identical to each other and to the complex crystallized in the space group C2 [J. Mol. Biol. 1992, 225, 445-456]. Actinomycin D intercalates into the middle sequence 5'-GC-3' from the minor groove of DNA. The two cyclic depsipeptide rings lie on both sides of the minor groove and cover the four base pairs of DNA. The drug is tightly connected to the DNA at the middle portion of the molecule by forming four threonine-guanine hydrogen bonds and two additional hydrogen bonds between the N2 amino group of phenoxazone and the DNA backbone. The four threonine-guanine hydrogen bonds appear to recognize the DNA sequence (5'-GC-3'). These essential hydrogen bonds are covered with the cyclic depsipeptides which are composed of mainly hydrophobic amino acid residues. However, careful examination of the structures indicates that three independent complex structures are significantly different but are rationally related. From the analysis, actinomycin D can easily change its conformation in order to fit its cyclic depsipeptides into the topographically different minor grooves of DNA's without breaking essential drug-DNA hydrogen bonds. These complex structures provide us with an additional insight into how the structure df actinomycin D has been effectively designed to bind tightly to various DNA. It is very rare that single crystal X-ray diffraction studies find three different modes for a drug to bind to the same DNA sequence. The structural data gained will be quite useful to interpret the multiple conformations of DNA-drug complexes observed by NMR studies and/or produced by the molecular mechanics and dynamics simulations. The sequence specificity of AMD is now rationally explained with the three different structures of the complex. Two independent structures of the host DNA, d(GAAGCTTC)(2), have also been determined by the X-ray diffraction method (cell dimension a = b = 70.54, c = 53.30 Angstrom; space group R3; R-factor = 0.212 at 3.0-Angstrom resolution). Both double-stranded DNA molecules adopt a canonical A-form DNA structure and have quite similar conformations. In both DNA's, the middle sequence, 5'-GC-3', which is the intercalation site of actinomycin D, is slightly unwound and opened up, suggesting that the host DNA takes an inherently favorable conformation for drug intercalation. A hypothetical binding process of actinomycin D has been proposed on the basis of the crystal structures of the DNA-actinomycin D complex and its host DNA. Simple molecular modeling and UV/vis spectroscopic studies have been utilized to further investigate the nature of actinomycin D. The results suggest that actinomycin D can bind intercalatively not only to B-form DNA but also to A-form DNA.
