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-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 +/- 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 A. 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.
Thorvaldsen
J L
JL
Department of Medicine, University of Utah Health Science Center, Salt Lake City 84132.
Sewell
A K
AK
Tanner
A M
AM
Peltier
J M
JM
Pickering
I J
IJ
George
G N
GN
Winge
D R
DR
D000595:Amino Acid Sequence; D001483:Base Sequence; D002175:Candida / Q000235:genetics*; D002942:Circular Dichroism; D003300:Copper / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins; D005800:Genes, Fungal; D005813:Genes, Synthetic; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013057:Spectrum Analysis; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014965:X-Rays; D015032:Zinc / Q000378:metabolism*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014157:Transcription Factors; C069807:AMT1 protein, Candida glabrate; D003300:Copper; D015032:Zinc