The role of intercalating residues in chromosomal high-mobility-group protein DNA binding, bending and specificity.
Ubiquitous high-mobility-group (HMGB) chromosomal proteins bind DNA in a non-sequence- specific fashion to promote chromatin function and gene regulation. Minor groove DNA binding of the HMG domain induces substantial DNA bending toward the major groove, and several interfacial residues contribute by DNA intercalation. The role of the intercalating residues in DNA binding, bending and specificity was systematically examined for a series of mutant Drosophila HMGB (HMG-D) proteins. The primary intercalating residue of HMG-D, Met13, is required both for high-affinity DNA binding and normal DNA bending. Leu9 and Tyr12 directly interact with Met13 and are required for HMG domain stability in addition to linear DNA binding and bending, which is an important function for these residues. In contrast, DNA binding and bending is retained in truncations of intercalating residues Val32 and Thr33 to alanine, but DNA bending is decreased for the glycine substitutions. Furthermore, substitution of the intercalating residues with those predicted to be involved in the specificity of the HMG domain transcription factors results in increased DNA affinity and decreased DNA bending without increased specificity. These studies reveal the importance of residues that buttress intercalating residues and suggest that features of the HMG domain other than a few base-specific hydrogen bonds distinguish the sequence-specific and non-sequence-specific HMG domain functions.
Klass
Janet
J
Department of Pharmacology, The University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262, USA.
Murphy
Frank V
FV
Fouts
Susan
S
Serenil
Melissa
M
Changela
Anita
A
Siple
Jessica
J
Churchill
Mair E A
ME
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000502:physiology; D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D006609:High Mobility Group Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
D000596:Amino Acids; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D006609:High Mobility Group Proteins; D004247:DNA