title: Comparative stability analysis of the thyroid transcription factor 1 and Antennapedia homeodomains: evidence for residue 54 in controlling the structural stability of the recognition helix.
abstract: The thyroid transcription factor 1 homeodomain (TTF-1 HD) shows a peculiar DNA-binding specificity which is partially dictated by several amino acids of the recognition helix. TTF-1 preferentially recognizes sequences containing the 5'-CAAG-3' core motif while most other homeodomains, such as Antennapedia (Antp), recognizes sites containing the 5'-TAAT-3' core motif. Since phenomena of 'induced fit' may occur during protein/DNA interaction, a primary role for high affinity binding and target discrimination has to be searched in the effect played by subtle structural determinants in these proteins. By using spectroscopic analysis in aqueous solution, we compared the structural stability of TTF-1 and Antp homeodomains. Although the three-dimensional structural architecture of homeodomains is conserved, some differences are detectable in terms of their structural stability. At 24 degrees C the TTF-1 HD is less structured than the Antp HD with 24 and 34% of the residues in the alpha-helical conformation, respectively. This poor folded structure reflects into different thermal and isothermal stability between the two homeodomains. TTF-1 HD exhibits a Tm of 39 degrees C and is stabilized by a delta GDH2O of +1487 cal/mol, calculated by Urea unfolding, while Antp HD exhibits a Tm of 48 degrees C and is stabilized by a delta GDH2O of +2742 cal/mol. By using mutants of both TTF-1 and Antp HDs we demonstrate that one of the major determinants in controlling the structural stability of the recognition helix is the residue at position 54. Since previous studies have shown that also residue at position 56 is involved in stabilization of the recognition helix, we conclude that the structure of this critical element is controlled by an interplay between residues at position 54 and 56 of the homeodomain.
authors.affiliation: Dipartimento di Scienze e Tecnologie Biomediche, Università degli Studi di Udine, Italy. GTell@makek.dstb.uniud.it
authors.forename: G
authors.initials: G
authors.lastname: Tell
authors.forename: R
authors.initials: R
authors.lastname: Acquaviva
authors.forename: S
authors.initials: S
authors.lastname: Formisano
authors.forename: F
authors.initials: F
authors.lastname: Fogolari
authors.forename: C
authors.initials: C
authors.lastname: Pucillo
authors.forename: G
authors.initials: G
authors.lastname: Damante
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D051943:Antennapedia Homeodomain Protein; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers / Q000235:genetics; D004355:Drug Stability; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics; D006358:Hot Temperature; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013961:Thyroid Gland / Q000378:metabolism; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics
chemical_list: D051943:Antennapedia Homeodomain Protein; D017931:DNA Primers; D018398:Homeodomain Proteins; C000614416:Nkx2-1 protein, rat; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors