Structure of the Binuclear Metal-Binding Site in the GAL4 Transcription Factor

The GAL4 transcription factor from yeast contains within its N-terminal DNA-binding domain an amino acid sequence containing six cysteine residues, C11-X₂-C14-X₆-C21-X₆-C28-X₂-C31-X₆-C38. The six Cys residues will form a binuclear metal cluster with either Zn(II) or Cd(II) in which two of the -S⁻ donors are bridging ligands between the two metal ions. Binding of Zn(II) or Cd(II) to the GAL4 DNA-binding domain is essential to induce the conformation of GAL4 required for the protein to recognize the specific DNA sequence, UAS_G, to which GAL4 binds. Evidence for the presence of the binuclear cluster has come from ¹¹³Cd NMR and 2D ¹H-¹¹³Cd heteronuclear NMR studies of the cloned DNA-binding domain of GAL4 consisting of the N-terminal 62 residues, GAL4(62*) [Pan and Coleman (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 2077]. Cd(II) binding to the GAL4 DNA is highly cooperative, thus the Cd₂Cys₆ cluster is always formed. On the other hand, Zn(II) forms well-defined Zn₁ and Zn₂ complexes with the DNA-binding domain of GAL4, both of which bind specifically to the UAS_G DNA sequence. The structural details of the Cd₂-, Zn₂-, and Zn, GAL4(62*) proteins have been determined by a variety of heteronuclear and 2D NMR techniques. When Cd(II) is exchanged for Zn(II), the cluster appears to expand to accommodate the larger Cd(II) ion as suggested by changes of 2 to 4 Hz in the ³J_HNα coupling constants for the amino acid residues which form the polypeptide loops enclosing the cluster, residues 10-40. These changes suggest alterations in the backbone φ torsional angles of from 20° to 30°. A metal-ligand structure derived from the ¹H-¹¹³Cd heteronuclear NMR as well as the polypeptide backbone connectivity around the cluster as determined from short-range ¹H-¹H NOE’s is presented. The metal ions also determine the major folding of GAL4(62*), since the chemical shift dispersion in the entire NH-αCH fingerprint region of the ¹H-¹H COSY spectrum collapses on removal of the metal ion. Two short segments of the GAL4(62*) polypeptide (residues 14-19 and 30-36 in the cluster forms, 12-19 and 30-36 in the Zn₁ species) show significant d/NN(i,i, +1) NOE’s. These short segments of polypeptide chain are the only ones that could be helical in the GAL4(62*). These NOE’s show significant differences in magnitude relative to the αN(i, i+1) NOE’s for the same residues between the Cd₂ and Zn₂ proteins as well as between the Zn₂ and Zn₁ proteins. Thus both the species of metal ion [Zn(II) or Cd(II)] and the number of metal ions bound determine the conformation of the peptide backbone.