Binding modes of distamycin A with d(CGCAAATTTGCG) sub 2 determined by two-dimensional NMR

Two-dimensional nuclear Overhauser effect spectroscopy (NOESY) was used to study the interaction of distamycin A with d(CGCAAATTTGCG){sub 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-DNA contact points, and to monitor exchange of the drug between binding sites. NOEs from drug pyrrole H3 to DNA C1{prime}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{prime}-AATT-3{prime} and 5{prime}-ATTT-3{prime} 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, to accommodate both drugs, indicating that the phosphate backbone can be distorted in response to ligand binding. Distance-constrained energy refinement of the 2:1 complexes indicates that electrostatic interactions, 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.