Use of a two-hybrid system to identify mutations in Max that confer increased affinity for Myc.
A yeast two-hybrid system was used to identify mutants of Max that exhibit an increased affinity for Myc. Truncated forms of the Max helix-loop-helix/leucine zipper motif (HLH/Zip) were first expressed in a two- hybrid system in which the bait protein was the HLH/Zip motif of Myc. Deletion of amino acids both amino-terminal and carboxy-terminal to the leucine zipper of Max reduced Myc/Max heterodimer formation as evidenced by a 160-fold reduction in the expression of the lacZ gene. A library of partially randomized sequences encoding this minimal leucine zipper of Max was then screened using the two-hybrid system. Mutant forms of the Max leucine zipper were identified whose affinities for Myc, as measured by beta-galactosidase activity in yeast lysates, were from 8- to 200-fold greater than the wild-type Max zipper. These Max mutants were shown to interact specifically with Myc and not with wild-type Max. Of 29 mutants analyzed, all had a unique amino acid sequence. This result illustrates the value of a genetic screen in the identification of a collection of mutant forms of the Max leucine zipper whose structures would not have been predicted based on principles of structure-based design.
Wang
H
H
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226, USA.
Van Den Bergh
F
F
Spencer
E
E
Wilcox
K
K
Herman
T
T
D001426:Bacterial Proteins / Q000235:genetics; D050976:Basic-Leucine Zipper Transcription Factors; D003062:Codon; D004268:DNA-Binding Proteins / Q000235:genetics*; D019281:Dimerization; D016259:Genes, myc / Q000235:genetics*; D005822:Genetic Vectors; D018257:Helix-Loop-Helix Motifs / Q000235:genetics*; D007763:Lac Operon; D016350:Leucine Zippers; D008957:Models, Genetic; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae; D012697:Serine Endopeptidases / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*; D015533:Transcriptional Activation; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D050976:Basic-Leucine Zipper Transcription Factors; D003062:Codon; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D000099160:LexA Repressor Protein; C067850:Myc associated factor X