Chemical shift mapped DNA-binding sites and N-15 relaxation analysis of the C-terminal KH domain of heterogeneous nuclear ribonucleoprotein K

The K homology (KH) motif is one of the major classes of nucleic acid bindi ng proteins. Some members of this family have been shown to interact with D NA while others have RNA targets. There have been no reports containing dir ect experimental evidence regarding the nature of KH module-DNA interaction . In this study, the interaction of the C-terminal KH domain of heterogeneo us nuclear ribonucleoprotein K (KH3) with it's cognate single-stranded DNA (ssDNA) are investigated. Chemical shift perturbation mapping indicates tha t the first two helices, the conserved GxxG loop, beta 1, and beta 2, are t he primary regions involved in DNA binding for KH3. The nature of the KH3-s sDNA interaction is further illuminated by a comparison of backbone N-15 re laxation data for the bound and unbound KH3. Relaxation data are also used to confirm that the backbone of wild-type KH3 is structurally identical to that of the G26R mutant KH3, which was previously published. Amide proton e xchange experiments indicate that the two helices involved in DNA binding a re less stable than other regions of secondary structure and that a large p ortion of KH3 backbone amide hydrogens are protected in some manner upon ss DNA binding. The major backbone dynamics features of KH3 are similar to the se of the structurally comparable human papillomavirus-31 E2 DNA binding do main. Secondary structure information for ssDNA-bound wild-type KH3 is also presented and shows that binding results in no global changes in the prote in fold.