NMR Scalar Couplings across Intermolecular Hydrogen Bonds between Zinc-Finger Histidine Side Chains and DNA Phosphate Groups

NMR scalar couplings across hydrogen bonds represent direct evidence for the partial covalent nature of hydrogen bonds and provide structural and dynamic information on hydrogen bonding. In this article, we report heteronuclear ¹⁵N-³¹P and ¹H-³¹P scalar couplings across the intermolecular hydrogen bonds between protein histidine (His) imidazole and DNA phosphate groups. These hydrogen-bond scalar couplings were observed for the Egr-1 zinc-finger-DNA complex. Although His side-chain NH protons are typically undetectable in heteronuclear ¹H-¹⁵N correlation spectra due to rapid hydrogen exchange, this complex exhibited two His side-chain NH signals around ¹H 14.3 ppm and ¹⁵N 178 ppm at 35 °C. Through various heteronuclear multidimensional NMR experiments, these signals were assigned to two zinc-coordinating His side chains in contact with DNA phosphate groups. The data show that the N_δ1 atoms of these His side chains are protonated and exhibit the ¹H-¹⁵N cross-peaks. Using heteronuclear ¹H, ¹⁵N, and ³¹P NMR experiments, we observed the hydrogen-bond scalar couplings between the His ¹⁵N_δ1/¹H_δ1 and DNA phosphate ³¹P nuclei. These results demonstrate the direct involvement of the zinc-coordinating His side chains in the recognition of DNA by the Cys₂His₂-class zinc fingers in solution.