Role of the zinc(II) ions in the structure of the three-finger DNA binding domain of the Sp1 transcription factor

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Abstract
The transcription factor Sp1 from Hela cells contains near the C-terminus of this protein of 778 amino acids three contiguous repeats of an amino acid sequence, -Cys-X4-Cys-X12-His-X3-His-, typical of the Cys2-His2-type zinc-finger DNA binding domain first found in transcription factor TFIIA. A DNA sequence corresponding to the codons from residue 614 to residue 779 of Sp1 (encompassing the three zinc-finger motifs) has been cloned and overproduced in Escherichia coli. The fragment of Sp1 containing the C-terminal 165 residues plus 2 from the cloning vector, designated Sp1 (167*), can be extracted with 5 M urea and then refolded in the presence of Zn(II) to a protein of specific conformation containing 3.0 .+-. 0.2 mol of tightly bound Zn(II)/mol of protein. Gel retardation assays using a labeled 14-bp DNA sequence containing a consensus Sp1 binding site show that the refolded Zn(ii) protein specifically recognizes the "GC box" sequence in the presence of a large excess of calf thymus DNA. Treatment of Zn(II)Sp1(167*) with 10 mM EDTA results in removal of Zn(II) and the formation of an apoprotein which does not specifically recognize DNA. Cd(II) can be exchanged for Zn(II)in the refolded protein with full retention of specific DNA recognition. This is the first Cys2His2-type "finger" protein where this substitution has been accomplished. Titration of the Zn(II) protein with 6 mol of p-mercuribenzenesulfonate/mol of protein results in the complete release of the three Zn(II) ions. Results of Zn(II) is highly cooperative. Reaction of only two of the sulfhydryl zinc ligands with the organic mercurial releases 75% of the Zn(II), suggesting that the Zn(II)-induced folding of the three fingers is probably cooprative. Circular dichroism shows the Zn(II)3 protein to contain .apprx.20% .alpha.-helix, .apprx.-20% .beta.-sheet, and .apprx.60% random coil as the secondary structure of the zinc-finger domain. A large part of the second structure is lost when the metal ions are removed.

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