Structural studies of the high mobility group globular domain and basic tail of HMG-D bound to disulfide cross-linked DNA.
HMG-D is an abundant high mobility group chromosomal protein present during early embryogenesis in Drosophila melanogaster. It is a non-sequence-specific member of a protein family that uses the HMG domain for binding to DNA in the minor groove. The highly charged C-terminal tail of HMG-D contains AK motifs that contribute to high-affinity non-sequence-specific DNA binding. To understand the interactions of the HMG domain and C-terminal tail of HMG-D with DNA in solution, a complex between a high-affinity truncated form of the protein and a disulfide cross-linked DNA fragment was studied using heteronuclear NMR techniques. Despite its relatively high affinity for the single "prebent" site on the DNA, K(d) = 1.4 nM, HMG-D forms a non-sequence-specific complex with the DNA as indicated by exchange broadening of the protein resonances at the DNA interface in solution. The secondary structural elements of the protein are preserved when the protein is complexed with the DNA, and the DNA-binding interface maps to the regions of the protein where the largest chemical shift differences occur. The C-terminal tail of HMG-D confers high-affinity DNA binding, has an undefined structure, and appears to make direct contacts in the major groove of DNA via residues that are potentially regulated by phosphorylation. We conclude that while the HMG domain of HMG-D recognizes DNA with a mode of binding similar to that used by the sequence-specific HMG domain transcription factors, there are noteworthy differences in the structure and interactions of the C-terminal end of the DNA-binding domain and the C-terminal tail.
Department of Pharmacology, University of Colorado Health Sciences Center, C236, 4200 East Ninth Avenue, Denver, Colorado 80262, USA.
L K
LK
Dow
D N
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Jones
S A
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Wolfe
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Verdine
M E
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Churchill
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002247:Carbon Isotopes; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D003903:Deuterium; D004331:Drosophila melanogaster; D006609:High Mobility Group Proteins / Q000737:chemistry*; D019476:Insect Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D002247:Carbon Isotopes; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D019476:Insect Proteins; D009587:Nitrogen Isotopes; C104923:high mobility group protein D; D004247:DNA; D003903:Deuterium