BINDING MODES OF DISTAMYCIN-A WITH D(CGCAAATTTGCG)2 DETERMINED BY 2-DIMENSIONAL NMR

Two-dimensional nuclear Overhauser effect spectroscopy (NOESY) was used to study the interaction of distamycin A with d(CGCAAATTTGCG)2. Spectra acquired at several points in a titration of the dodecamer with distamycin A were used to assign resonances, to determine drug-drug and drug-DNAcontact points, and to monitor exchange of the drug between binding sites. At low drug:DNA ratios (0.5 equiv), both one-drug and symmetric two-drug binding modes were observed, while at high ratios (2 equiv), the two-drug complex was the primary species present. The off-rate for the drug from the 2:1 mode was found to be slow on the NMR time scale (0.2 Ñ 0.1 s-1, 30°C), facilitating characterization of the distamycin A binding sites in this mode. NOEs from drug pyrrole H3 to DNA Cl-H and adenine C2H protons, as well as observed line width changes of the DNA protons as a function of temperature, were consistent with a model in which two drugs bind simultaneously, overlapping in the minor groove, with each drug sliding between 5'-AATT-3' and 5'-ATTT-3' binding sites at a rate fast on the NMR time scale. Molecular modeling of the 2:1 complexes indicates that the minor groove must expand, relative to the 1:1 complex, toaccommodate both drugs, indicating that the phosphate backbone can be distorted in response to ligand binding. Distance-constrained energy refinement of the 2:1complexes indicates that electrostaticinteractions, hydrogen bonds between the drugs and the DNA, and both drug-drug and drug-DNA stacking interactions all contribute to stabilization of the complex. Comparisons are made with crystallographic studies of this drug and dodecamer. Implications of the 2:1 binding mode for other studies and possibilities for the design of new sequence-specific recognition complexes are discussed. © 1990, American Chemical Society. All rights reserved.