title: Folding topology of the disulfide-bonded dimeric DNA-binding domain of the myogenic determination factor MyoD.
abstract: The myogenic determination factor MyoD is a member of the basic-helix-loop-helix (bHLH) protein family. A 68-residue fragment of MyoD encompassing the entire bHLH region (MyoD-bHLH) is sufficient for protein dimerization, sequence-specific DNA binding in vitro, and conversion of fibroblasts into muscle cells. The circular dichroism spectrum of MyoD-bHLH indicates the presence of significant alpha-helical secondary structure; however, the NMR spectrum lacks features of a well-defined tertiary structure. There is a naturally occurring cysteine at residue 135 in mouse MyoD that when oxidized to a disulfide induces MyoD-bHLH to form a symmetric homodimer with a defined tertiary structure as judged by sedimentation equilibrium ultracentrifugation and NMR spectroscopy. Oxidized MyoD-bHLH retains sequence-specific DNA-binding activity, albeit with an apparent 100-1000-fold decrease in affinity. Here, we report the structural characterization of the oxidized MyoD-bHLH homodimer by NMR spectroscopy. Our findings indicate that the basic region is unstructured and flexible, while the HLH region consists of two alpha-helices of unequal length connected by an as yet undetermined loop structure. Qualitative examination of interhelical NOEs suggests several potential arrangements for the two helix 1/helix 2 pairs in the symmetric oxidized dimer. These arrangements were evaluated for whether they could incorporate the disulfide bond, satisfy loop length constraints, and juxtapose the two basic regions. Only a model that aligns helix 1 parallel to helix 1' and antiparallel to helix 2 was consistent with all constraints. Thus, an antiparallel four-helix bundle topology is proposed for the symmetric dimer. This topology is hypothesized to serve as a general model for other bHLH protein domains.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle 98195.
authors.forename: M A
authors.initials: MA
authors.lastname: Starovasnik
authors.forename: T K
authors.initials: TK
authors.lastname: Blackwell
authors.forename: T M
authors.initials: TM
authors.lastname: Laue
authors.forename: H
authors.initials: H
authors.lastname: Weintraub
authors.forename: R E
authors.initials: RE
authors.lastname: Klevit
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004926:Escherichia coli; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000378:metabolism; D017570:MyoD Protein; D010084:Oxidation-Reduction; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014461:Ultracentrifugation
chemical_list: D004268:DNA-Binding Proteins; D004220:Disulfides; D046911:Macromolecular Substances; D009124:Muscle Proteins; D017570:MyoD Protein; D011994:Recombinant Proteins; D004247:DNA