Oxygen-to-Sulfur Substitution of DNA Phosphate Entropically Enhances Protein-DNA Affinity

Abstract

For some proteins, dithioation of DNA phosphate is known to enhance binding affinities. For the Antennapedia (Antp) homedomain - DNA complex, we have mechanistically characterized this phenomenon by integrated use of fluorescence, isothermal calorimetry (ITC), NMR spectroscopy, and X-ray crystallography. Through ITC and fluorescence, we found that this affinity enhancement is entropy-driven. By NMR, we investigated the ionic hydrogen bonds and internal motions of lysine side-chain NH3+ groups involved in ion pairs with DNA. By X-ray crystallography, we compared the structures of complexes with and without dithioation of the phosphate. Our NMR and X-ray data demonstrate that the lysine side chain in contact with the DNA phosphate becomes more dynamic upon dithioation. Our thermodynamic, structural, and dynamic investigations collectively show that the affinity enhancement by the oxygen-to-sulfur substitution in DNA phosphate is largely due to an entropic gain arising from mobilization of the intermolecular ion pair at the protein-DNA interface.This work was supported by Grant R01-GM105931 from the National Institutes of Health (to J.I.) and Grant CHE-1307344 from the National Science Foundation (to J.I.).