Scaloni A, Monti M, Acquaviva R, Tell G, Damante G, Formisano S, Pucci P
Centro Internazionale di Servizi di Spettrometria di Massa, CNR-Università di Napoli "Federico II", Italy.
Biochemistry. 1999 Jan 5;38(1):64-72. doi: 10.1021/bi981300k.
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.
通过一种将有限蛋白酶解和选择性化学修饰实验与质谱方法相结合的策略，对甲状腺转录因子1同源结构域（TTF-1HD）-DNA复合物的拓扑结构进行了研究。当在不存在或存在其靶寡核苷酸的情况下用该蛋白质进行有限蛋白酶解消化时，获得了差异肽图，从中可以推断出参与相互作用的氨基酸残基。同样，对分离的和复合的同源结构域中的赖氨酸残基进行选择性乙酰化，使我们能够鉴定出因与DNA相互作用而受到保护的氨基酸。在中性pH下对分离的TTF-1HD进行的表面拓扑分析与在酸性条件下通过核磁共振研究确定的分子三维结构高度一致。在蛋白质的C末端区域检测到微小差异，与核磁共振数据相反，该区域对蛋白酶没有可及性。对复合物的分析为基于与迄今描述的同源结构域结构的同源性提出的模型提供了实验验证。相互作用后观察到C末端区域的可及性增加，表明其被寡核苷酸分子从蛋白质核心中置换出来。对具有不同序列和结合能力的DNA片段进行的比较实验突出了复合物之间的结构差异，主要位于同源结构域的N末端区域，从而解释了它们不同的解离常数。
