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

Authors

  • James C. Hu,

    1. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
    Current affiliation:
    1. Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843
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  • Robert T. Sauer,

    1. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
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  • Nicholas E. Newell,

    1. Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, Massachusetts 02142
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  • Bruce Tidor

    Corresponding author
    1. Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, Massachusetts 02142
    • Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, Massachusetts 02142
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Abstract

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 α-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 λ 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.

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