NMR structure determination reveals that the homeodomain is connected through a flexible linker to the main body in the Drosophila Antennapedia protein.
The secondary structure of an N-terminally elongated Antennapedia (Antp) homeodomain (HD) polypeptide containing residues -14 to 67, where residues 1-60 constitute the HD, has been determined by NMR in solution. This polypeptide contains the conserved motif -Tyr-Pro-Trp-Met- (YPWM) at positions -9 to -6. Despite the hydrophobic nature of this tetrapeptide motif, the N-terminal arm consisting of residues -14 to 6 is flexibly disordered, and the well-defined part of the HD structure with residues 7-59 is indistinguishable from that of the shorter Antp HD polypeptide (where positions 0, 1, and 67 are methionine, arginine, and glycine, respectively). In vitro biochemical studies showed that the stability and specificity of the DNA binding previously observed for the shorter Antp HD polypeptide is preserved in the elongated polypeptide. These results strongly support the view that the HD is connected through a flexible linker to the main body in the Antp protein and that the minor groove contacts by the N-terminal arm (residues 1-6) in the Antp HD-DNA complex are an intrinsic feature of the DNA-binding interactions of the intact Antp protein.
Qian
Y Q
YQ
Institut für Molekularbiologie und Biophysik, Eidgenössiche Technische Hochschule-Hönggerberg, Zürich, Switzerland.
Otting
G
G
Furukubo-Tokunaga
K
K
Affolter
M
M
Gehring
W J
WJ
Wüthrich
K
K
D000595:Amino Acid Sequence; D000818:Animals; D051943:Antennapedia Homeodomain Protein; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004330:Drosophila / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins; D005801:Genes, Homeobox; D018398:Homeodomain Proteins; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors
D051943:Antennapedia Homeodomain Protein; C497974:Antp protein, Drosophila; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011522:Protons; D011994:Recombinant Proteins; D014157:Transcription Factors; D004247:DNA