title: Design of a "minimAl" homeodomain: the N-terminal arm modulates DNA binding affinity and stabilizes homeodomain structure.
abstract: This report investigates the sequence specificity requirements for homeodomain structure and DNA binding activity by the design and synthesis of a "minimAl" homeodomain (for minimalist design and alanine scanning mutagenesis) which contains the consensus residues and in which all nonconsensus residues have been replaced with alanine. The murine homeodomain Msx served as the prototype for the minimAl homeodomain, Ala-Msx. We show that Ala-Msx binds to DNA specifically, albeit with lower affinity than Msx. A derivative of the minimAl homeodomain, Ala-Msx(NT), which contains a native rather than an alanine-substituted N-terminal arm, has similar DNA binding affinity as Msx. We show that the native N-terminal arm stabilizes the tertiary structure of the minimAl homeodomain. Although Ala-Msx resembles a molten-globule protein, the structure of Ala-Msx(NT) is similar to Msx. The requirement for an intact N-terminal arm is not unique to the minimAl homeodomain, since the N-terminal arm also promotes high-affinity binding activity and appropriate tertiary structure of Msx. Therefore, the homeodomain "scaffold" consists of consensus residues, which are sufficient for DNA recognition, and nonconsensus residues in the N-terminal arm, which are required for optimal DNA binding affinity and appropriate tertiary structure. MinimAl design provides a powerful strategy to probe homeodomain structure and function. This approach should be of general utility to study the sequence specificity requirements for structure and function of other DNA-binding domains.
authors.affiliation: Department of Neuroscience and Cell Biology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway.
authors.forename: Z
authors.initials: Z
authors.lastname: Shang
authors.forename: V E
authors.initials: VE
authors.lastname: Isaac
authors.forename: H
authors.initials: H
authors.lastname: Li
authors.forename: L
authors.initials: L
authors.lastname: Patel
authors.forename: K M
authors.initials: KM
authors.lastname: Catron
authors.forename: T
authors.initials: T
authors.lastname: Curran
authors.forename: G T
authors.initials: GT
authors.lastname: Montelione
authors.forename: C
authors.initials: C
authors.lastname: Abate
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D016384:Consensus Sequence; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry*; D005801:Genes, Homeobox; D018398:Homeodomain Proteins; D051957:MSX1 Transcription Factor; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014157:Transcription Factors
chemical_list: D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D051957:MSX1 Transcription Factor; D011994:Recombinant Proteins; D014157:Transcription Factors; D000409:Alanine