Artificial zinc finger peptide containing a novel his₄ domain

Zinc finger constitutes one of the most common DNA binding motifs. Although zinc finger proteins consisting of Cys₂His₂, Cys₃His, Cys₄, and Cys₆ domains are known in nature, a novel His₄ zinc finger protein has never been observed. Herein, we have created the first artificial His₄-type zinc finger protein (H₄Sp1) engineered by Cys → His mutations of the Cys₂His₂-type zinc finger transcription factor Sp1. The CD features of the single finger H₄Sp1f2 and three-finger H₄Sp1 clearly demonstrate the folding of the mutant His₄ peptides by complexation with Zn(II). The NMR study of Zn(II)-H₄Sp1f2 reveals that some distortions of the helical region occur due to Zn(II) coordination. The gel mobility shift assay and DNase I footprinting analysis strongly show the binding of Zn(II)-H₄Sp1 to the GC-box site of duplex DNA. The methylation interference pattern of Zn(II)-H₄Sp1 binding significantly resembles that of the corresponding C₂H₂Sp1 binding. The present artificial peptide H₄Sp1 is the first example of a zinc finger containing the His₄ domain. Of special interest is the fact that the zinc finger domains of H₄Sp1 are folded (although not identical to the native structure) and bind DNA similar to wild-type C₂H₂Sp1.