Probing the roles of residues at the e and g positions of the GCN4 leucine zipper by combinatorial mutagenesis.

Combinatorial mutagenesis with an alphabet limited to alanine, glutamic acid, lysine, and threonine was used to probe the role of interactions involving surface residues in stabilizing a short alpha-helical coiled coil. The residues at eight e and g positions in the leucine zipper of the Saccharomyces cerevisiae transcription factor GCN4 were randomized to these four residues in a lambda repressor-leucine zipper fusion protein, resulting in 65,536 possible residue combinations. Roughly three-fourths of these combinations allowed stable coiled-coil formation as assayed by DNA binding by the fusion protein. To understand the basis for the activity differences, functional and non-functional mutants were sequenced and statistical tests were applied to identify structure/function correlations. Helix-forming propensity and favorable intrasubunit and intersubunit charge-charge interactions were positively correlated with activity. These studies suggest that the identities of surface side chains at the e and g positions of coiled coils contribute 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.