Identification of the Zn(II) site in the copper-responsive yeast transcription factor, AMT1: a conserved Zn module.
The N-terminal metal-binding domains of the copper-activated yeast transcription factors, ACE1 and AMT1, bind to specific DNA sequences in a Cu-dependent fashion. Recombinant AMT1 and ACE1 metal-binding domains are isolated as Cu4Zn1-protein complexes. Site-directed mutagenesis of AMT1 was used in this study to map the ligands of the Cu(I) and Zn(II) ions. The results are consistent with the N-terminal halves of AMT1 and ACE1 consisting of two independent submodules, one binding a single Zn(II) ion and the second binding the tetracopper cluster. The basis of this conclusion is, first, that mutations of two cysteinyl codons and a histidyl codon in the first 42 residues of AMT1 do not alter DNA binding. In contrast, serine substitutions at four cysteine positions at codons 43, 61, 90, and 98 abolish DNA binding. We demonstrated previously that population of the Zn(II) site in AMT1 does not alter the ability of the protein to bind DNA but bound Cu(I) ions are essential for DNA binding [Thorvaldsen, J. L., et al. (1994) Biochemistry 33, 9566-9577]. Second, mutations in the N-terminal 42 residue segment reduce the Zn(II) content of purified mutant AMT1 molecules. Third, a synthetic peptide consisting of the N-terminal 42 residues in AMT1 forms a stable Zn(II) complex and substitution with Co(II) reveals an electronic spectrum identical to that of the Co-substituted intact Cu4AMT1 protein. 113Cd(II) NMR studies reveal that the divalent metal site consists of ligands provided by three cysteinyl thiolates and a single histydyl imidazole. The sequence homology between AMT1, ACE1, and MAC1 in the N-terminal 42 residues suggests that ACE1 and MAC1 will, likewise, contain N-terminal Zn modules. A 42-residue ACE1 synthetic peptide gives identical metal binding properties to the corresponding AMT1 synthetic peptide. Thus, AMT1 and likely ACE1 consist of two contiguous modules, residues 1-42 forming an independent Zn(II) module and residues 43-110 enfolding a tetracopper cluster.
Farrell
R A
RA
University of Utah Health Sciences Center, Salt Lake City 84132, USA.
Thorvaldsen
J L
JL
Winge
D R
DR
D000595:Amino Acid Sequence; D001665:Binding Sites; D002175:Candida / Q000235:genetics; D017124:Conserved Sequence; D003300:Copper / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins; D008024:Ligands; D008667:Metalloproteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
C060256:CUP2 protein, S cerevisiae; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D008024:Ligands; D008667:Metalloproteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C069807:AMT1 protein, Candida glabrate; D003300:Copper; D015032:Zinc