High affinity DNA-binding Myc analogs: recognition by an alpha helix.
Myc and other basic-helix-loop-helix-leucine zipper (b-HLH-ZIP) proteins bind the sequence CACGTG. Exhaustive mutagenesis in the basic domain identified four amino acids critical for DNA binding with spacing suggestive of an alpha-helical face. Surprisingly, two highly conserved amino acids were nonessential for DNA binding. Circular dichroism demonstrated a DNA-induced alpha-helical transition. A series of analogs were constructed with multiple simultaneous alanine substitutions at nonessential positions and a critical lysine for arginine substitution. In this way 35-fold higher specific affinity for CACGTG was obtained as compared with the basic domain of c-Myc. These b-HLH-ZIP proteins appear to bind the same palindromic sequence and may compete for common sites in vivo. Additionally, a C-terminal basic region clamp motif was identified that was also identifiable in crystal structures from several different families of DNA-binding factors.
Fisher
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Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
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Sharp
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D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D017433:Protein Structure, Secondary; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D009838:Oligodeoxyribonucleotides; D016271:Proto-Oncogene Proteins c-myc; C064972:TFEB protein, human; D014157:Transcription Factors; D000409:Alanine