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. 1989 May;86(9):3145-9.
doi: 10.1073/pnas.86.9.3145.

Structure and function of the Zn(II) binding site within the DNA-binding domain of the GAL4 transcription factor

T Pan  1 , J E Coleman
Affiliations

Structure and function of the Zn(II) binding site within the DNA-binding domain of the GAL4 transcription factor

T Pan et al. Proc Natl Acad Sci U S A. 1989 May.

Abstract

The transcription factor GAL4 from Saccharomyces cerevisiae contains a "zinc-finger"-like motif, Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-Cys-Xaa2-Cys-Xaa6+ ++-Cys, within its DNA-binding domain. A GAL4 fragment consisting of residues 1-147 plus two additional residues from the cloning vector [denoted GAL4(149*)] has been cloned and overexpressed in Escherichia coli. This fragment includes the entire DNA-binding domain (residues 1-74). The homogeneous GAL4-(149*) protein contains 1-1.5 moles of Zn(II) per mole of protein. The GAL4(149*) protein binds tightly to the specific 17-base-pair palindromic DNA sequence found at GAL4 binding sites as shown by gel-retention assays using a 32P-labeled 23-mer containing this sequence. Removal of the intrinsic Zn(II) by EDTA at low pH abolishes binding to the 23-mer. The GAL4(149*) apoprotein can be reconstituted with Zn(II), Cd(II), or Co(II) with restoration of specific DNA binding. Titration of GAL4(149*) apoprotein with 113Cd(II) shows two 113Cd(II) binding sites on the molecule, one with delta of 707 ppm, suggesting coordination to four sulfur atoms, and one with delta of 669 ppm, suggesting coordination to three or four sulfur atoms. Because GAL4(149*) protein contains only six cysteine residues within its DNA-binding domain, the precise coordination of the two Cd(II) ions cannot be stated with certainty; one or more shared -S- ligands could exist. GAL4(149*) protein contains approximately 40% alpha-helix and approximately 20% beta-sheet, estimated from circular dichroism. Removal of the native Zn(II) ion causes limited unfolding of secondary structure, but less than one turn of alpha-helix. The binding of Zn(II), Cd(II), and, to a lesser extent, Co(II) to GAL4(149*) apoprotein protects the protein from proteolysis by trypsin, which produces a 13-kDa DNA-binding core.

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