Department of Chemistry and Biochemistry, Florida International University, Miami, 33199, USA.
Biomolecular Sciences Institute, Florida International University, Miami, 33199, USA.
Nucleic Acids Res. 2022 Mar 21;50(5):2431-2439. doi: 10.1093/nar/gkac115.
The mammalian high mobility group protein AT-hook 2 (HMGA2) houses three motifs that preferentially bind short stretches of AT-rich DNA regions. These DNA binding motifs, known as 'AT-hooks', are traditionally characterized as being unstructured. Upon binding to AT-rich DNA, they form ordered assemblies. It is this disordered-to-ordered transition that has implicated HMGA2 as a protein actively involved in many biological processes, with abnormal HMGA expression linked to a variety of health problems including diabetes, obesity, and oncogenesis. In the current work, the solution binding dynamics of the three 'AT-hook' peptides (ATHPs) with AT-rich DNA hairpin substrates were studied using DNA UV melting studies, fluorescence spectroscopy, native ion mobility spectrometry-mass spectrometry (IMS-MS), solution isothermal titration calorimetry (ITC) and molecular modeling. Results showed that the ATHPs bind to the DNA to form a single, 1:1 and 2:1, 'key-locked' conformational ensemble. The molecular models showed that 1:1 and 2:1 complex formation is driven by the capacity of the ATHPs to bind to the minor and major grooves of the AT-rich DNA oligomers. Complementary solution ITC results confirmed that the 2:1 stoichiometry of ATHP: DNA is originated under native conditions in solution.
哺乳动物的高迁移率族蛋白 A 类蛋白 2（HMGA2）含有三个基序，这些基序优先结合富含 AT 的短 DNA 区域。这些 DNA 结合基序被称为“AT 钩”，传统上被认为是无结构的。与富含 AT 的 DNA 结合后，它们形成有序的组装。正是这种无序到有序的转变使 HMGA2 成为一种积极参与许多生物学过程的蛋白质，异常的 HMGA 表达与多种健康问题有关，包括糖尿病、肥胖症和肿瘤发生。在当前的工作中，使用 DNA UV 熔融研究、荧光光谱学、天然离子迁移谱-质谱（IMS-MS）、溶液等温滴定量热法（ITC）和分子建模研究了三个“AT 钩”肽（ATHPs）与富含 AT 的 DNA 发夹底物的结合动力学。结果表明，ATHPs 与 DNA 结合形成单一的、1:1 和 2:1 的“键锁”构象集合。分子模型表明，1:1 和 2:1 复合物的形成是由 ATHPs 结合富含 AT 的 DNA 寡聚物的小沟和大沟的能力驱动的。补充的溶液 ITC 结果证实，ATHP:DNA 的 2:1 计量比是在溶液中原位条件下产生的。
