MIXED CU-BINDING DOMAIN OF THE AMT1 TRANSCRIPTION FACTOR FROM CANDIDA-GLABRATA( AND ZN2+ COORDINATION IN THE DNA)

AMT1 is the transcription factor required for Cu-induced expression of metallothionein genes in the yeast Candida glabrata. The copper-bindi ng, DNA-binding domain of AMT 1 has been purified after expression of an AMT1 synthetic gene in bacteria and was confirmed as active in a ge l shift assay. The Cu-activated AMT1 was shown to contain a Cu+-thiola te tetracopper center and a single Zn2+ site. AMT1 is purified as a Cu -Zn protein from bacterial cultures grown in the presence of CuSO4. Ch emical analysis suggested that 4.2 +/- 0.2 and 1.2 +/- 0.2 molar equiv copper and zinc ions bound, respectively. Electrospray mass spectrome try was used to verify that a uniform species was present with 4 Cu+ i ons and 1 Zn2+ ion bound per AMT1 molecule. Cu+ binding to form a tetr acopper center occurs cooperatively as shown by electrospray MS of apo AMT 1 samples reconstituted with increasing equivalency of Cu+. Copper -thiolate coordination was indicated by Cu-S charge-transfer transitio ns in the ultraviolet, luminescence typical of Cu-thiolate clusters an d EXAFS. Analysis of the EXAFS of CuZnAMT1 revealed predominantly trig onal Cu+ coordination and the presence of a polycopper cluster by virt ue of a short Cu-Cu distance of 2.7 Angstrom. Zn K-edge EXAFS of Cu(4) Zn(1)AMT1 and electronic spectroscopy of AMT 1 with Co2+ substituted f or the single Zn2+ ion are consistent with tetrahedral Zn2+ coordinati on with thiolate ligands. The Cu-activated AMT1 exhibited a conformati on distinct from that of metal-free AMT1 as shown by circular dichrois m. DNA binding by AMT1 was dependent on the tetracopper center but was independent of occupancy of the Zn2+ site. This is the first report o f a single, uniform tetracopper center in a metal-activated transcript ion factor.