MULTIPLE BINDING MODES OF ANTICANCER DRUG ACTINOMYCIN-D - X-RAY, MOLECULAR MODELING, AND SPECTROSCOPIC STUDIES OF D(GAAGCTTC)(2)-ACTINOMYCIN-D COMPLEXES AND ITS HOST DNA

The antitumor drug actinomycin D intercalates into DNA and strongly in hibits RNA transcription at very low concentration. Two independent st ructures of d(GAAGCTTC)(2)-actinomycin D complexes have been determine d: 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 basicall y identical to each other and to the complex crystallized in the space group C2 [J. Mol. Biol. 1992, 225, 445-456]. Actinomycin D intercalat es 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 a nd 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 threonin e-guanine hydrogen bonds and two additional hydrogen bonds between the N2 amino group of phenoxazone and the DNA backbone. The four threonin e-guanine hydrogen bonds appear to recognize the DNA sequence (5'-GC-3 '). These essential hydrogen bonds are covered with the cyclic depsipe ptides which are composed of mainly hydrophobic amino acid residues. H owever, careful examination of the structures indicates that three ind ependent complex structures are significantly different but are ration ally related. From the analysis, actinomycin D can easily change its c onformation in order to fit its cyclic depsipeptides into the topograp hically different minor grooves of DNA's without breaking essential dr ug-DNA hydrogen bonds. These complex structures provide us with an add itional insight into how the structure df actinomycin D has been effec tively designed to bind tightly to various DNA. It is very rare that s ingle crystal X-ray diffraction studies find three different modes for a drug to bind to the same DNA sequence. The structural data gained w ill be quite useful to interpret the multiple conformations of DNA-dru g complexes observed by NMR studies and/or produced by the molecular m echanics and dynamics simulations. The sequence specificity of AMD is now rationally explained with the three different structures of the co mplex. Two independent structures of the host DNA, d(GAAGCTTC)(2), hav e 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 a t 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 intercalati on site of actinomycin D, is slightly unwound and opened up, suggestin g that the host DNA takes an inherently favorable conformation for dru g intercalation. A hypothetical binding process of actinomycin D has b een proposed on the basis of the crystal structures of the DNA-actinom ycin D complex and its host DNA. Simple molecular modeling and UV/vis spectroscopic studies have been utilized to further investigate the na ture of actinomycin D. The results suggest that actinomycin D can bind intercalatively not only to B-form DNA but also to A-form DNA.