Mutagenesis analysis of the interaction between the dorsal rel homology domain and HMG boxes of DSP1 protein.
DSP1 is an HMG-like protein of Drosophila melanogaster consisting of 386 amino acids with two HMG domains at the C-terminal end. It was shown to interact with Dorsal protein through the HMG domains and to enhance its DNA binding. Each HMG domain consists of approximately 80 amino acid residues, forming three alpha helices folded into an L-shaped structure. We have compared the interaction of various truncated and mutated forms of DSP1 with the dorsal Rel homology domain (RHD). In particular, we have mutated the conserved tryptophan residue 212 or 302 in A or B boxes or the lysine-rich region ((253)KKRK(256)) of the A/B linker. Analysis by circular dichroism revealed that the protein tertiary structure is affected in these mutants. However, these mutations do not abolish the DSP1 binding to Dorsal, except if the two HMG boxes are altered, i.e., in a double mutant or in mutant isolated domain. Finally, studies on the enhancement of Dorsal DNA binding by DSP1 revealed that the DNA affinity is maximum in the presence of wild-type DSP1, is dramatically reduced when box A is altered, and is completely abolished when box B is altered.
Martin
Davy
D
Centre de Biophysique Moléculaire, CNRS, conventionné avec l'Université d'Orléans, rue Charles Sadron, 45071 Orléans cedex 2, France.
Daulny
Anne
A
Decoville
Martine
M
Locker
Daniel
D
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics*; D004331:Drosophila melanogaster; D004591:Electrophoresis, Polyacrylamide Gel; D006609:High Mobility Group Proteins / Q000737:chemistry* / Q000235:genetics*; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics*; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics*; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics*; D014364:Tryptophan / Q000737:chemistry
C104348:DSP1 protein, Drosophila; D029721:Drosophila Proteins; D006609:High Mobility Group Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C062236:dl protein, Drosophila; D014364:Tryptophan; D004247:DNA; D008239:Lysine