title: Chemical shift mapped DNA-binding sites and 15N relaxation analysis of the C-terminal KH domain of heterogeneous nuclear ribonucleoprotein K.
abstract: The K homology (KH) motif is one of the major classes of nucleic acid binding proteins. Some members of this family have been shown to interact with DNA while others have RNA targets. There have been no reports containing direct experimental evidence regarding the nature of KH module-DNA interaction. In this study, the interaction of the C-terminal KH domain of heterogeneous nuclear ribonucleoprotein K (KH3) with its cognate single-stranded DNA (ssDNA) are investigated. Chemical shift perturbation mapping indicates that the first two helices, the conserved GxxG loop, beta 1, and beta 2, are the primary regions involved in DNA binding for KH3. The nature of the KH3-ssDNA interaction is further illuminated by a comparison of backbone 15N relaxation 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 exchange experiments indicate that the two helices involved in DNA binding are less stable than other regions of secondary structure and that a large portion of KH3 backbone amide hydrogens are protected in some manner upon ssDNA binding. The major backbone dynamics features of KH3 are similar to those of the structurally comparable human papillomavirus-31 E2 DNA binding domain. Secondary structure information for ssDNA-bound wild-type KH3 is also presented and shows that binding results in no global changes in the protein fold.
authors.affiliation: Laboratory of Biophysical Chemistry, Building 3, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-0380, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Baber
authors.forename: D
authors.initials: D
authors.lastname: Levens
authors.forename: D
authors.initials: D
authors.lastname: Libutti
authors.forename: N
authors.initials: N
authors.lastname: Tjandra
mesh_terms: D000577:Amides; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D017354:Point Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000235:genetics; D011522:Protons; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D000577:Amides; D004268:DNA-Binding Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009587:Nitrogen Isotopes; D009841:Oligonucleotides; D011522:Protons; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins