Conformational dynamics in specialized C₂H₂zinc finger domains enable zinc-responsive gene repression in S. pombe

Abstract

Loz1 is a zinc-responsive transcription factor in fission yeast that maintains cellular zinc homeostasis by repressing the expression of genes required for zinc uptake in high zinc conditions. Previous deletion analysis of Loz1 found a region containing two tandem C2H2zinc-fingers and an upstream "accessory domain" rich in histidine, lysine, and arginine residues to be sufficient for zinc-dependent DNA binding and gene repression. Here we report unexpected biophysical properties of this pair of seemingly classical C2H2zinc fingers. Isothermal titration calorimetry and NMR spectroscopy reveal two distinct zinc binding events localized to the zinc fingers. NMR spectra reveal complex dynamic behavior in this zinc responsive region spanning time scales from fast 10-12-10-10to slow > 100 sec. Slow exchange due to cis-trans isomerization of the TGERP linker results in doubling of many signals in the protein. Conformational exchange on the 10-3s timescale throughout the first zinc finger distinguishes it from the second and is linked to weaker affinity for zinc. These findings reveal the mechanism of zinc sensing by Loz1 and illuminate how the protein's rough free-energy landscape enable zinc sensing, DNA binding and regulated gene expression.