Jamshidi, C. | Search bioRxiv

bioRxiv2024

Conformational dynamics in specialized C2H2 zinc finger domains enable zinc-responsive gene repression in S. pombe

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 C2H2 zinc-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 C2H2 zinc 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 10-12-10-10 to 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 10-3 s 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 proteins rough free-energy landscape enable zinc sensing, DNA binding and regulated gene expression. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/614115v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@8bb7ddorg.highwire.dtl.DTLVardef@d43153org.highwire.dtl.DTLVardef@f25930org.highwire.dtl.DTLVardef@bd7c8b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Insights into Ligand-Mediated Activation of an Oligomeric Ring-Shaped Gene-Regulatory Protein from Solution- and Solid-State NMR

The 91 kDa oligomeric ring-shaped ligand binding protein TRAP (trp RNA binding attenuation protein) regulates the expression of a series of genes involved in tryptophan (Trp) biosynthesis in bacilli. When cellular Trp levels rise, the free amino acid binds to sites buried in the interfaces between each of the 11 (or 12, depending on the species) protomers in the ring. Crystal structures of Trp-bound TRAP show the Trp ligands are sequestered from solvent by a pair of loops from adjacent protomers that bury the bound ligand via polar contacts to several threonine residues. Binding of the Trp ligands occurs cooperatively, such that successive binding events occur with higher apparent affinity but the structural basis for this cooperativity is poorly understood. We used solution methyl-TROSY NMR relaxation experiments focused on threonine and isoleucine sidechains, as well as magic angle spinning solid-state NMR 13C-13C and 15N-13C chemical shift correlation spectra on uniformly labeled samples recorded at 800 and 1200 MHz, to characterize the structure and dynamics of the protein. Methyl 13C relaxation dispersion experiments on ligand-free apo TRAP revealed concerted exchange dynamics on the {micro}s-ms time scale, consistent with transient sampling of conformations that could allow ligand binding. Cross-correlated relaxation experiments revealed widespread disorder on fast timescales. Chemical shifts for methyl-bearing side chains in apo- and Trp-bound TRAP revealed subtle changes in the distribution of sampled sidechain rotameric states. These observations reveal a pathway and mechanism for induced conformational changes to generate homotropic Trp-Trp binding cooperativity.

biophysics↗