Topology of the Thyroid Transcription Factor 1 Homeodomain−DNA Complex

Abstract

The topology of the thyroid transcription factor 1 homeodomain (TTF-1HD)-DNA complex was investigated by a strategy which combines limited proteolysis and selective chemical modification experiments with mass spectrometry methodologies. When limited proteolysis digestions were carried out with the protein in the absence or presence of its target oligonucleotide, differential peptide maps were obtained from which the amino acid residues involved in the interaction could be inferred. Similarly, selective acetylation of lysine residues in both the isolated and the complexed homeodomain allowed us to identify the amino acids protected by the interaction with DNA. Surface topology analysis of isolated TTF-1HD performed at neutral pH was in good agreement with the three-dimensional structure of the molecule as determined by NMR studies under acidic conditions. Minor differences were detected in the C-terminal region of the protein which, contrary to NMR data, showed no accessibility to proteases. Analysis of the complex provided an experimental validation of the model proposed on the basis of the homology with the homeodomain structures described so far. An increased accessibility of the C-terminal region was observed following the interaction, suggesting its displacement from the protein core by the oligonucleotide molecule. Comparative experiments with DNA fragments differing in sequence and binding capabilities highlighted structural differences among the complexes, mainly located in the N-terminal region of the homeodomain, thus accounting for their different dissociation constants.

Probing protein structure by amino acid‐specific covalent labeling and mass spectrometry

“…Consequently, covalent labeling of lysine residues along with MS detection has been used effectively to study protein-nucleic acid interactions, which are particularly important to regulate DNA replication and transcription. The surface topology of the thyroid transcription factor 1 homeodomain (TTF1HD)-DNA complex was characterized by Scaloni et al who used a combination of limited proteolysis, selective acetylation with acetic anhydride, and MS analysis (Scaloni et al, 1999). MALDI-TOF MS and limited proteolysis revealed that the N-terminus, Lys4, and Lys25 were acetylated in the isolated protein but not in the TTF1-HD-oligonucleotide complex; that difference indicated that there is a structural change in these regions upon interaction with DNA.…”

Characterization of transcription factors by mass spectrometry and the role of SELDI‐MS

“…A similar study probed the topology of the thyroid transcription-factor 1 homeodomain by limited proteolysis and chemical modification experiments combined with electrospray ionization-MS (ESI-MS) and MALDI (Scaloni et al, 1999). Limited proteolysis of the apo-form and of the DNA-bound transcription factor, in addition to lysine-acetylation protection experiments, provided a structural model that was consistent with the known 3-dimensional structure previously obtained by NMR.…”

Molecular analysis of a human PAX6 homeobox mutant

“…37,38 We have also successively used this kind of approach to study the topology of a HD containing transcription factor (ie TTF-1) bound to DNA 39 and found strong similarities with data obtained by NMR studies. 40 It is known that trypsin cleaves at the C-terminal sides of both the Lys and Arg residues of proteins; therefore, we speculated that mutations affecting Arg residues would alter the trypsin proteolytic pattern of the PAX6 mutant.…”