Influence of amino acid numbers between two ligand cysteines of zinc finger proteins on affinity and specificity of DNA binding.
The C(2)H(2)-type zinc finger motif has the consensus sequence X(2)-Cys-X(2,4)-Cys-X(12)-His-X(3-5)-His and two or four amino acid residues between two ligand cysteines are well conserved. To evaluate this conservation, we prepared six mutant peptides derived from the three-zinc-finger domain of Sp1 whose C-terminal finger has two amino acid residues between the two invariant cysteines. Their circular dichroism spectra suggest that these mutants have an ordered secondary structure comparable with that of the wild type. The insertion mutants (X=3-5) bind to DNA with the somewhat lower affinity than the wild type (X=2). On the other hand, the DNA binding affinity of the deletion mutants (X=0 and 1) is significantly reduced. In particular, the extent of the reduction in two mutants with Cys-Cys and Cys-Pro-Cys is remarkable. The methylation interference analyses demonstrate that this decreased DNA binding affinity is due to lowering of the extent of the base contacts by the central and C-terminal fingers. This information reveals not only a clue to evolution of the zinc finger but also the possibility of the existence of the zinc finger with altered number of amino acid residues between the two ligand cysteines.
Nagaoka
Makoto
M
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
Kondo
Yumi
Y
Uno
Yumiko
Y
Sugiura
Yukio
Y
D000818:Animals; D001665:Binding Sites; D016384:Consensus Sequence; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D019175:DNA Methylation; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D016329:Sp1 Transcription Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers
D008024:Ligands; D010455:Peptides; D016329:Sp1 Transcription Factor; D004247:DNA; D003545:Cysteine