Solution structure of the DNA-binding domain of Cd₂-GAL4 from S. cerevisiae

Abstract

THE GAL4 protein activates transcription of the genes required for galactose utilization in Saccharomyces cerevisiae¹. The protein, consisting of 881 amino acids, is dimeric when bound to one of the approximately twofold symmetrical DNA sites present in the galactose upstream activating sequence (UAS_G)^2–5. Here we use two-dimensional NMR spectroscopy to determine the structure of an amino-terminal fragment of GAL4 (residues 1-65). This fragment, a monomer in solution, binds as a dimer specifically to UAS_G-containing DNA. Residues 9-40 form a well defined, compact globular cluster, whereas residues 1-8 and 41-66 show considerable conformational mobility in the absence of DNA. The compact domain contains a motif in which six cysteines, located on two symmetrically related helix/extended strand units connected by a long loop, coordinate two central zinc ions, forming a bimetal-thiolate cluster^6–11. The zincs were replaced by NMR-active¹¹³Cd in most of our work and structural parameters are therefore derived from the Cd₂-protein. The structure obtained for the GAL4 DNA-binding domain represents a novel DNA-binding motif. Essentially the same conformation is observed for the compact domain in solution using NMR techniques as was seen for the central core of the N-terminal fragment bound to DNA using crystallographic techniques¹². Thus, the core of the DNA-binding domain changes little upon binding DNA.