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 th e formation of a tetracopper-thiolate cluster. Recombinant Amt1 (resid ues 1-110) is isolated as a Cu,ZnAmt1 complex. Previous mapping studie s [Farrell et al, (1996) Biochemistry 35, 1571-1580] revealed that the Zn(II) site is enfolded by an independent, N-terminal domain consisti ng 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 asa C uAmt1 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) io ns bound in the 110-mer Amt1 molecule in the energies and intensities of the ultraviolet S --> Cu charge transfer transitions and luminescen ce. Copper K-edge extended X-ray absorption fine structure spectroscop y (EXAFS) of the truncated CuAmt1 complex revealed the same 2.26 Angst rom mean Cu-S bond distance as in the Cu,ZnAmt1 complex. A diagnostic feature of the polycopper-thiolate cluster in Cu,ZnAmt1 is the short 2 .7 Angstrom Cu-Cu distance determined by Cu K-edge EXAFS. The truncate d CuAmt1 complex had the same short 2.7 Angstrom Cu-Cu distance. The t runcated 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 ad jacent Cu regulatory domain (residues 41-110), and a C-terminal transc riptional activation domain. Cu(I) activation of Amt1 appears to consi st of conversion of the 70-residue Cu regulatory domain from an inacti ve conformer to a structure containing the tetracopper cluster.