title: Probing the roles of residues at the e and g positions of the GCN4 leucine zipper by combinatorial mutagenesis.
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 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.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
authors.forename: J C
authors.initials: JC
authors.lastname: Hu
authors.forename: N E
authors.initials: NE
authors.lastname: Newell
authors.forename: B
authors.initials: B
authors.lastname: Tidor
authors.forename: R T
authors.initials: RT
authors.lastname: Sauer
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics*; D014644:Genetic Variation; D005971:Glutamates / Q000235:genetics; D018698:Glutamic Acid; D005998:Glycine / Q000235:genetics; D016350:Leucine Zippers / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011494:Protein Kinases / Q000096:biosynthesis / Q000235:genetics*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013223:Statistics as Topic; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D005971:Glutamates; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D018698:Glutamic Acid; D011494:Protein Kinases; D005998:Glycine