Nuclear magnetic resonance spectroscopy of a DNA complex with the uniformly 13C-labeled Antennapedia homeodomain and structure determination of the DNA-bound homeodomain.
A 1:1 complex formed by the mutant Antennapedia(C39S) homeodomain and a 14 base-pair DNA duplex (molecular weight approximately 18,000) was prepared in which the protein was uniformly 13C-labeled. Using two-dimensional nuclear Overhauser enhancement (NOE) spectroscopy with 13C(omega 1, omega 2)-double-half-filter and three-dimensional 13C-correlated NOE spectroscopy, nearly complete sequence-specific resonance assignments were obtained for both the protein and the DNA in the complex. On this basis conformational constraints needed for a three-dimensional structure determination were collected. Using 855 intramolecular distance constraints as input, the structure of the DNA-bound Antp(C39S) homeodomain was calculated with the program DIANA, followed by restrained energy minimization with the program OPAL. A group of 20 conformers characterizes a well-defined structure for residues 8 to 56, with an average of 0.5 A of the pairwise root-mean-square deviations calculated for the backbone atoms of the individual conformers relative to the mean coordinates. The quality of the resulting structure is comparable to the one for the free protein, and the global fold of the free Antp(C39S) homeodomain was found to be conserved in the DNA complex. The structure of the DNA-bound protein was the starting point for the subsequent structure determination of the complete Antp(C39S) homeodomain-DNA complex in solution.
Qian
Y Q
YQ
Institut für Molekularbiologie und Biophysik Eidgenössische Technische Hochschule-Hönggerberg, Zürich, Switzerland.
Otting
G
G
Billeter
M
M
Müller
M
M
Gehring
W
W
Wüthrich
K
K
D000595:Amino Acid Sequence; D000818:Animals; D051943:Antennapedia Homeodomain Protein; D004268:DNA-Binding Proteins / Q000737:chemistry*; D003852:Deoxyribonucleoproteins / Q000737:chemistry*; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D005801:Genes, Homeobox; D018398:Homeodomain Proteins; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins; D014157:Transcription Factors
D051943:Antennapedia Homeodomain Protein; C497974:Antp protein, Drosophila; D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors