Oligomerization properties of GCN4 leucine zipper e and g position mutants.
Putative intersubunit electrostatic interactions between charged amino acids on the surfaces of the dimer interfaces of leucine zippers (g-e' ion pairs) have been implicated as determinants of dimerization specificity. To evaluate the importance of these ionic interactions in determining the specificity of dimer formation, we constructed a pool of > 65,000 GCN4 leucine zipper mutants in which all the e and g positions are occupied by different combinations of alanine, glutamic acid, lysine, or threonine. The oligomerization properties of these mutants were evaluated based on the phenotypes of cells expressing lambda repressor-leucine zipper fusion proteins. About 90% of the mutants do not form stable homooligomers. Surprisingly, approximately 8% of the mutant sequences have phenotypes consistent with the formation of higher-order (> dimer) oligomers, which can be classified into three types based on sequence features. The oligomerization states of mutants from two of these types were determined by characterizing purified fusion proteins. The Type I mutant behaved as a tetramer under all tested conditions, whereas the Type III mutant formed a variety of higher-order oligomers, depending on the solution conditions. Stable homodimers comprise less than 3% of the pool; several g-e' positions in these mutants could form attractive ion pairs. Putative repulsive ion pairs are not found among the homodimeric mutants. However, patterns of charged residues at the e and g positions do not seem to be sufficient to predict either homodimer or heterodimer formation among the mutants.
Zeng
X
X
Department of Biochemistry & Biophysics, Texas A&M University, College Station 77843-2128, USA.
Zhu
H
H
Lashuel
H A
HA
Hu
J C
JC
D000595:Amino Acid Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D019281:Dimerization; D004563:Electrochemistry; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D016350:Leucine Zippers; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D011494:Protein Kinases