Solution Structure of the Head-to-Head Dimer of Calicheamicin Oligosaccharide Domain and d(CGTAGGATATCCTACG)₂

The head-to-head dimer of the calicheamicin oligosaccharide domain exhibits an impressive nanomolar affinity for its specific DNA recognition sites and a substantially higher degree of sequence selectivity relative to the oligosaccharide monomer. In an effort to determine the structural basis for these binding properties, the solution structure of the 1:1 complex between the head-to-head dimer and the self-complementary oligonucleotide d(CGTAGGATATCCTACG)₂ has been solved using ¹H NMR-derived distance and torsion angle constraints and molecular dynamics calculations. Complete sequence specific proton assignments of both the DNA duplex and the carbohydrate have been obtained by 2D-NMR. A total of 607 experimentally derived constraints were identified including 452 proton−proton distance constraints derived from NOESY cross peaks intensities and assigned hydrogen bonds, along with 155 dihedral angle constraints obtained from a detailed analysis of the multiplet structure of cross-peaks for the sugar rings and from qualitative analysis of nuclear Overhauser effects for the DNA backbone. The final conformation of the complex is represented by an ensemble of seven structures (the average all-atom root mean square deviation from the mean is 1.07 Å in the well-defined region) obtained by refining 14 initial conformations with widely different nonstandard DNA geometries. A number of favorable interactions are found to stabilize the structure of the complex and account for binding sequence preferences. Overall, the binding mode of each oligosaccharide unit of the head-to-head dimer in the DNA minor groove seems to be very close to that observed in the case of the monomeric calicheamicin oligosaccharide bound to its corresponding TCCT recognition site. Variable temperature NMR studies have shown that this dimer binds to d(CGTAGGATATCCTACG)₂ in two subtly different conformations, probably differing in the positioning of rings E and E‘, interconverting with a rate constant of ∼0.35 s^-1. The solution structure of this carbohydrate−DNA complex provides confirmation of design principles for new calicheamicin-based DNA-binding agents and confirms insights obtained previously into the molecular basis for oligosaccharide recognition within the DNA minor groove.