Alpha-helical linker of an artificial 6-zinc finger peptide contributes to selective DNA binding to a discontinuous recognition sequence.
Although many zinc finger motifs have been developed to recognize specific DNA triplets, a rational way to selectively skip a particular non-recognized gap in the DNA sequence has never been established. We have now created a 6-zinc finger peptide with an alpha-helix linker, Sp1ZF6(EAAAR)4, which selectively binds to the discontinuous recognition sites in the same phase (10 bp gap) against the opposite phase (5 bp gap) of the DNA helix. The linker peptide (EAAAR)4 forms an alpha-helix structure stabilized by salt bridges, and the helical length is estimated to be about 30 A, corresponding to that of the 10 bp DNA. The gel shift assays demonstrate that Sp1ZF6(EAAAR)4 preferably binds to the 10 bp-gapped target rather than the 5 bp-gapped target. The CD spectra show that the alpha-helical content of the (EAAAR)4 linker is higher in the complex with the 10 bp-gapped target than in the complex with the 5 bp-gapped target. The present results indicate that the alpha-helical linker is suitable for binding to the recognition sites in the same phase and that the linker induces the loss of binding affinity to the recognition sites with the opposite phase. The engineering of a helix-structured linker in the 6-zinc finger peptides should be one of the most promising approaches for selectively targeting discontinuous recognition sites depending on their phase situations.
Yan
Wei
W
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
Imanishi
Miki
M
Futaki
Shiroh
S
Sugiura
Yukio
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000138:chemical synthesis / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D016329:Sp1 Transcription Factor / Q000737:chemistry / Q000378:metabolism; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D010455:Peptides; D016329:Sp1 Transcription Factor; C523121:Sp1ZF6(EAAAR)4 zinc finger protein; C523122:Sp1ZF6(G4S)4 zinc finger protein; C523123:Sp1ZF6(KAAEA)4 zinc finger protein; D004247:DNA