Presence of a copper(I)-thiolate regulatory domain in the copper-activated transcription factor Amt1.

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. -->
Grant Information: ES 03817 United States ES NIEHS NIH HHS; SP30-CA 42014 United States SP CSAP SAMHSA HHS
Substance Nomenclature: 0 (DNA-Binding Proteins)
0 (Fungal Proteins)
0 (Transcription Factors)
141490-28-0 (AMT1 protein, Candida glabrate)
789U1901C5 (Copper)
9007-49-2 (DNA)
Entry Date(s): Date Created: 19961119 Date Completed: 19970107 Latest Revision: 20131121
Update Code: 20240829
DOI: 10.1021/bi961642v
PMID: 8931556
Autor: Graden JA; University of Utah Health Sciences Center, Salt Lake City 84132, USA., Posewitz MC, Simon JR, George GN, Pickering IJ, Winge DR
Jazyk: angličtina
Zdroj: Biochemistry [Biochemistry] 1996 Nov 19; Vol. 35 (46), pp. 14583-9.
DOI: 10.1021/bi961642v
Abstrakt: 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.
Databáze: MEDLINE
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