Presence of a copper(I)-thiolate regulatory domain in the copper-activated transcription factor Amt1.
The Amt1 transcription factor from Candida glabrata is activated by the formation of a tetracopper-thiolate cluster. Recombinant Amt1 (residues 1-110) is isolated as a Cu,ZnAmt1 complex. Previous mapping studies [Farrell et al. (1996) Biochemistry 35, 1571-1580] revealed that the Zn(II) site is enfolded by an independent, N-terminal domain consisting of residues 1-40. One prediction from the mapping study is that the tetracopper cluster is enfolded by residues 41-110. A truncated Amt1 peptide consisting of residues 37-110 was expressed and isolated as a CuAmt1 complex with 4 mol equiv of Cu(I) bound. The bound Cu(I) ions in the truncated Amt1 complex were spectroscopically similar to Cu(I) ions bound in the 110-mer Amt1 molecule in the energies and intensities of the ultraviolet S-->Cu charge transfer transitions and luminescence. Copper K-edge extended X-ray absorption fine structure spectroscopy (EXAFS) of the truncated CuAmt1 complex revealed the same 2.26 A mean Cu-S bond distance as in the Cu,ZnAmt1 complex. A diagnostic feature of the polycopper-thiolate cluster in Cu,-ZnAmtl1 is the short 2.7 A Cu-Cu distance determined by Cu K-edge EXAFS. The truncated CuAmt1 complex had the same short 2.7 A Cu-Cu distance. The truncated CuAmt1 complex bound DNA specifically and with high affinity consistent with residues 41-110 being an independent domain stabilized by the tetracopper cluster. Thus, Amt1 consists of three independent and contiguous domains, an N-terminal Zn module (residues 1-40), an adjacent Cu regulatory domain (residues 41-110), and a C-terminal transcriptional activation domain. Cu(I) activation of Amt1 appears to consist of conversion of the 70-residue Cu regulatory domain from an inactive conformer to a structure containing the tetracopper cluster.
Graden
J A
JA
University of Utah Health Sciences Center, Salt Lake City 84132, USA.
Posewitz
M C
MC
Simon
J R
JR
George
G N
GN
Pickering
I J
IJ
Winge
D R
DR
D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D003300:Copper; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins; D008969:Molecular Sequence Data; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D014157:Transcription Factors; C069807:AMT1 protein, Candida glabrate; D003300:Copper; D004247:DNA