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-speci fic 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 A K motifs that contribute to high-affinity non-sequence-specific DNA binding . To understand the interactions of the HMG domain and C-terminal tail of H MG-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 p rotein resonances at the DNA interface in solution. The secondary structura l 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 w here the largest chemical shift differences occur. The C-terminal tail of H MG-D confers high-affinity DNA binding, has an undefined structure, and app ears to make direct contacts in the major groove of DNA via residues that a re 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 notew orthy differences in the structure and interactions of the C-terminal end o f the DNA-binding domain and the C-terminal tail.