title: Interconversion between serine and aspartic acid in the alpha helix of the N-terminal zinc finger of Sp1: implication for general recognition code and for design of novel zinc finger peptide recognizing complementary strand.
abstract: In the typical base recognition mode of the C(2)H(2)-type zinc finger, the amino acid residues at alpha-helical positions -1, 3, and 6 make a contact with the base in one strand (the primary strand), and the residue at position 2 interacts with the base in a complementary strand (the secondary strand). The N-terminal zinc finger of the three-zinc-finger domain of Sp1 has inherently a unique five-base-pair binding mode in which the guanine bases are recognized in both strands. To clarify the effect of the amino acid at position 2 on DNA binding affinity and base specificity, we have created a library of the mutants by the interconversion between serine and aspartic acid in the N-terminal zinc finger of Sp1 and recombinant variants of finger order. Gel mobility shift and methylation interference assays showed that the combination of arginine and serine at positions -1 and 2, respectively, provides a newly strong guanine contact in the secondary strand and a higher binding affinity than that of wild-type Sp1. Of special interest are the facts that the mutant with lysine and aspartic acid at positions -1 and 2 in the alpha helix predominantly recognizes the bases in the secondary strand and that its DNA binding affinity is higher than that of the wild-type. The aspartic acid or serine at position 2 independently contributes to the DNA binding affinity and base specificity. The present results provide useful information for the design of a novel zinc finger protein with priority for the bases in the secondary strand.
authors.affiliation: Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Nagaoka
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Shiraishi
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Uno
authors.forename: Wataru
authors.initials: W
authors.lastname: Nomura
authors.forename: Yukio
authors.initials: Y
authors.lastname: Sugiura
mesh_terms: D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine; D016329:Sp1 Transcription Factor / Q000737:chemistry*; D016335:Zinc Fingers
chemical_list: D011994:Recombinant Proteins; D016329:Sp1 Transcription Factor; D001224:Aspartic Acid; D012694:Serine; D004247:DNA