The three-dimensional structure of the vnd/NK-2 homeodomain-DNA complex by NMR spectroscopy.
The three-dimensional solution structure obtained by NMR of the complex formed between the uniformly singly15N and doubly13C/15N-labeled vnd/NK-2 homeodomain and its consensus 16 base-pair DNA binding sequence was determined. This work was carried out using the accepted repertoire of experiments augmented with a novel implementation of the water flipback technique to enhance signals from exchangeable amide protons. The results using this new technique confirm the existence of hydrogen bonding between the invariant Asn51 and the second adenine of the DNA binding sequence, as seen in crystal structures of other homeodomain-DNA complexes, but never before detected by NMR. Hydrogen bonding by Arg5 and Lys3 in the minor groove of the DNA appears to be responsible for two unusually upfield-shifted ribose H1' resonances. The DNA duplex is nearly straight and its structure is primarily that of B -DNA. A detailed comparison is presented for all available homeodomain-DNA structures including the vnd/NK-2 DNA complex, which demonstrates that homology is maintained in the protein structure, whereas for the orientation of the homeodomain relative to DNA, small but significant variations are observed. Interactions are described involving certain residues in specific positions of the homeodomain, namely Leu7, Thr41, and Gln50 of vnd/NK-2, where single amino acid residue mutations lead to dramatic developmental alterations. The availability of our previously determined three- dimensional structure of the vnd/NK-2 homeodomain in the absence of DNA allows us to assess structural changes in the homeodomain induced by DNA binding.
Gruschus
J M
JM
Laboratory of Biophysical Chemistry, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20982-0380, USA.
Tsao
D H
DH
Wang
L H
LH
Nirenberg
M
M
Ferretti
J A
JA
D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D029721:Drosophila Proteins; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000378:metabolism*; D007089:Image Enhancement; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012996:Solutions; D014157:Transcription Factors
D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D012996:Solutions; D014157:Transcription Factors; C094797:vnd protein, Drosophila; D004247:DNA