Mixed Cu+ and Zn2+ Coordination in the DNA-Binding Domain of the AMT1 Transcription Factor from Candida glabrata

AMT1 is the transcription factor required for Cu-induced expression of metallothionein genes in the yeast Candida glabrata. The copper-binding, DNA-binding domain of AMT1 has been purified after expression of an AMT1 synthetic gene in bacteria and was confirmed as active in a gel shift assay. The Cu...

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Bibliographic Details
Published in:Biochemistry (Easton) 1994-08, Vol.33 (32), p.9566-9577
Main Authors: Thorvaldsen, Joanne L, Sewell, Andrew K, Tanner, Amie M, Peltier, John M, Pickering, Ingrid J, George, Graham N, Winge, Dennis R
Format: Article
Language:English
Subjects:
ADN
Amino Acid Sequence
Base Sequence
Candida - genetics
CINC
Circular Dichroism
COBRE
Copper - metabolism
CUIVRE
DNA-Binding Proteins - genetics
DNA-Binding Proteins - metabolism
Escherichia coli - genetics
Fungal Proteins
Genes, Fungal
Genes, Synthetic
Mass Spectrometry
Molecular Sequence Data
Peptide Fragments - genetics
Peptide Fragments - metabolism
Protein Binding
PROTEINAS
PROTEINE
Recombinant Proteins - metabolism
Spectrophotometry, Ultraviolet
Spectrum Analysis
TORULOPSIS
Transcription Factors - genetics
Transcription Factors - metabolism
X-Rays
ZINC
Zinc - metabolism
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Summary:AMT1 is the transcription factor required for Cu-induced expression of metallothionein genes in the yeast Candida glabrata. The copper-binding, DNA-binding domain of AMT1 has been purified after expression of an AMT1 synthetic gene in bacteria and was confirmed as active in a gel shift assay. The Cu-activated AMT1 was shown to contain a Cu+-thiolate tetracopper center and a single Zn2+ site. AMT1 is purified as a Cu-Zn protein from bacterial cultures grown in the presence of CuSO4. Chemical analysis suggested that 4.2 plus 0.2 and 1.2 +/- 0.2 molar equiv copper and zinc ions bound, respectively. Electrospray mass spectrometry was used to verify that a uniform species was present with 4 Cu+ ions and 1 Zn2+ ion bound per AMT1 molecule. Cu+ binding to form a tetracopper center occurs cooperatively as shown by electrospray MS of apoAMT1 samples reconstituted with increasing equivalency of Cu+. Copper-thiolate coordination was indicated by Cu-S charge-transfer transitions in the ultraviolet, luminescence typical of Cu-thiolate clusters and EXAFS. Analysis of the EXAFS of CuZnAMT1 revealed predominantly trigonal Cu+ coordination and the presence of a polycopper cluster by virtue of a short Cu-Cu distance of 2.7 angstroms. Zn K-edge EXAFS of Cu4Zn1AMT1 and electronic spectroscopy of AMT1 with Co2+ substituted for the single Zn2+ ion are consistent with tetrahedral Zn2+ coordination with thiolate ligands. The Cu-activated AMT1 exhibited a conformation distinct from that of metal-free AMT1 as shown by circular dichroism. DNA binding by AMT1 was dependent on the tetracopper center but was independent of occupancy of the Zn2+ site. This is the first report of a single, uniform tetracopper center in a metal-activated transcription factor
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00198a024