NMR studies of the DNA-binding domain of ADR1

Thesis (Ph. D.)--University of Washington, 1996Nuclear magnetic resonance spectroscopy was used to study the DNA-binding domain of yeast transcription factor ADR1. A polypeptide containing the minimal DNA-binding region was overexpressed in E. coli and purified to homogeneity. The construct, ADR1z, contains two Cys$\sb2$-His$\sb2$ zinc fingers and an N-terminal sequence required for tight DNA binding. Resonance assignments are presented for ADR1z free and bound to specific DNA. The assignment strategy involved traditional NOESY methods, specific amino acid labeling, and triple resonance experiments. Structural data are presented in the form of chemical shift perturbation maps, $\sp{13}$C chemical shift index analyses, NOE connectivities, and $\rm\sp3J\sb{HN\alpha}$ coupling constants. The zinc fingers of ADR1z differ little in structure from single finger peptides and do not interact in the absence of DNA. The zinc fingers do not alter structure upon DNA binding. In contrast, the N-terminal flanking sequence is random coil in the absence of DNA. This region becomes less mobile, less exposed to solvent, and more structured upon DNA binding. The N-terminus does not interact with exposed portions of the zinc fingers in the complex and likely enhances binding by contacting the DNA directly. No fully regular pattern of secondary structure is detected for the bound N-terminus. This region is not helical; it probably adopts a largely extended conformation. The orientation of the N-terminus with respect to the DNA site is deduced from perturbations induced by fixed paramagnetic cobalt. The N-terminus doubles back and runs antiparallel to the zinc fingers, approaching the center of the palindrome. Chemical shift perturbations are used to map the protein-DNA interface. In striking agreement with previous mutagenesis experiments, these data suggest that contacts arise from R115, H118, and R121 of finger 1 and R143 of finger 2. The fingers bind DNA in different orientations, with the entire helix of finger 1 but only the extreme N-terminus of the helix of finger 2 in proximity to DNA. Suboptimal contact by finger 2 likely necessitates the unusual third contact of finger 1 and the participation of the N-terminus in DNA binding