Solution Structure of a Zap1 Zinc-responsive Domain Provides Insights into Metalloregulatory Transcriptional Repression in Saccharomyces cerevisiae

The Zap1 transcription factor controls expression of genes that regulate zinc homeostasis in Saccharomyces cerevisiae. The solution structure of two zinc fingers (zf1-2CA3) derived from a zinc-responsive domain of Zap1 (zf1-2) has been determined. Under zinc-limiting conditions, zinc finger 2 (zf2) from this domain has been shown to be a constitutive transcriptional activator. Moreover, repression of zf2 function in zinc-replete cells required zinc coordination to both canonical finger 1 (zf1) and zf2 metal sites, suggesting zf1–zf2 cooperativity underlies Zap1 metalloregulation. A structural basis for this cooperativity is identified here. Favorable inter-helical contacts in zf1-2CA3 extend the individual finger hydrophobic cores through the zf1–zf2 interface. Tryptophan residues at position 5 in each finger provide numerous non-helical inter-finger contacts reminiscent of those observed in GLI1 zinc fingers 1 and 2. The molecular mechanism for zf1-dependent repression of zf2 transcriptional activation is explored further using NMR and CD titration studies. While zf1 independently forms a ββα solution structure, the majority of zf2 ensemble solution states do not adopt the canonical ββα zinc finger fold without zf1–zf2 interactions. Cooperative effects on Zn(II) affinities stemming from these finger–finger interactions are observed also in calorimetric studies, in which the 160(±20) nM (zf1) and 250(±40) nM (zf2) Kd values for each individual finger increased substantially in the context of the zf1-2 protein (apparent Kdzf1-2WT=4.6(±1.2) nM). On the basis of the above observations, we propose a mechanism for Zap1 transcriptional regulation in which zf1–zf2 interactions stabilize the ββα folded “repressed state” of the zf2 activation domain in the presence of cellular Zn(II) excess. Moreover, in contrast to earlier reports of ≪1 labile zinc ion/Escherichia coli cell, the zf1–zf2 zinc affinities determined calorimetrically are consistent with Zn(II) levels ≫1 labile zinc ion/eukaryotic cell.