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, UASG, 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 UASG DNA sequence. The structural details of the Cd2-, Zn2-, and Zn1GAL4(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 3JHN alpha 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 phi torsional angles of from 20 degrees to 30 degrees. 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-alpha CH 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 dNN(i,i + 1) NOE's. These short segments of polypeptide chain are the only ones that could be helical in the GAL4(62*).(ABSTRACT TRUNCATED AT 400 WORDS)

• PMID: 1958667
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Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, Non-P.H.S. | Research Support, U.S. Gov't, P.H.S.

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Gardner KH, Pan T, Narula S, Rivera E, Coleman JE

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