PROBING THE ROLES OF RESIDUES AT THE E-POSITION AND G-POSITION OF THEGCN4 LEUCINE-ZIPPER BY COMBINATORIAL MUTAGENESIS

Combinatorial mutagenesis with an alphabet limited to alanine, glutami c acid, lysine, and threonine was used to probe the role of interactio ns involving surface residues in stabilizing a short alpha-helical coi led coil. The residues at eight e and g positions in the leucine zippe r of the Saccharomyces cerevisiae transcription factor GCN4 were rando mized to these four residues in a lambda repressor-leucine zipper fusi on protein, resulting in 65,536 possible residue combinations. Roughly three-fourths of these combinations allowed stable coiled-coil format ion as assayed by DNA binding by the fusion protein. To understand the basis for the activity differences, functional and nonfunctional muta nts were sequenced and statistical tests were applied to identify stru cture/function correlations. Helix-forming propensity and favorable in trasubunit and intersubunit charge-charge interactions were positively correlated with activity. These studies suggest that the identities o f surface side chains at the e and g positions of coiled coils contrib ute modestly to stability; by comparison with previous work, however, the e and g positions are far less critical than residues at the a and d positions, which form the hydrophobic core of the dimer interface.