title: The N-terminal methionine is a major determinant of the DNA binding specificity of MEF-2C.
abstract: Members of the MEF-2 family of transcriptional regulators positively modulate the activity of basic helix-loop-helix proteins in both myogenic and neurogenic cell lineages. Previous work had shown that MEF-2C(2-117), a protein fragment comprising the dimerization and DNA-binding domains of MEF-2C but lacking the N-terminal methionine, bound to AT-rich DNA sequences with high affinity. MEF-2C(2-117) did not discriminate between different AT-rich sequences. We now report the in vitro DNA binding properties of a MEF-2C fragment containing the N-terminal methionine. Measurements of the apparent dissociation constants of the complexes of GG-MEF-2C(1-117) revealed that different AT-rich sequences are bound with different affinities; in particular MEF site containing DNA (CTATAAATAG) is bound preferentially to DNA containing a SRF site (CATAAATG). Strikingly, when the shorter AT run consisted of six alternating thymines and adenines, almost wild-type affinity was observed. Irrespective of the particular DNA sequence, all circular dichroism spectra of the DNA complexes of GG-MEF-2C(1-117) were superimposable and characterized by an identical maximal ellipticity at 269.5 nm, suggesting similar DNA conformations. Bending analysis by circular permutation assay revealed that on complex formation MEF-2C(2-117) induced cognate DNA to bend by 49 degrees, while heterologous DNA remained unbent. In the presence of the N-terminal methionine, however, all DNA sequences were bent by 70 degrees. The above results suggest an important function for the N-terminal methionine in properly orientating MEF-2C on the DNA.
authors.affiliation: Department of Chemistry, ETH-Zürich, CH-8092 Zurich, Switzerland.
authors.forename: D
authors.initials: D
authors.lastname: Meierhans
authors.forename: R K
authors.initials: RK
authors.lastname: Allemann
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D005786:Gene Expression Regulation / Q000235:genetics; D064326:MEF2 Transcription Factors; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D018006:Myogenic Regulatory Factors / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding / Q000235:genetics; D013816:Thermodynamics
chemical_list: D004268:DNA-Binding Proteins; D064326:MEF2 Transcription Factors; D018006:Myogenic Regulatory Factors; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D004247:DNA; D008715:Methionine