Three-dimensional Structures Of Biological Macromolecule

生物大分子三维结构

• 批准号: 7154350
• 负责人: Bernard R Brooks
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7154350
• 关键词: DNA binding protein; Drosophilidae; arthropod genetics; binding sites; gene mutation; genetically modified animals; homeobox genes; macromolecule; nuclear magnetic resonance spectroscopy; nucleic acid sequence; nucleic acid structure; peptide structure; protein binding; protein sequence; site directed mutagenesis; structural biology

Using molecular modeling and NMR spectroscopy, the three-dimensional structures of wild type and mutant homeodomain containing proteins in the NK-2 class, the human CSX/NKX-2.5, and NKX-3.1 proteins, and the unique HOP homeodomain, have been investigated in the free state, bound to DNA, and in ternary complexes with associated transcription factors. The most recent results focus on the structural mechanisms of DNA specificity and enhancer complex assembly, including interaction of human Nkx3.1, CSX/NKX-2.5 and HOP with serum response factor (SRF). NMR of these homeoproteins have revealed novel interaction motifs and suppression mechanisms playing crucial roles in cardiogenesis, which have subsequently been confirmed by functional assays with mutations in the regions identified by NMR. Investigation of the human homeodomain T41M mutant of Nkx3.1, an oncogene found to co-segregate with prostate cancer risk, showed that the homeodomain was thermally destabilized, though its structure and DNA binding were otherwise unaltered. NMR and calorimetry studies comparing the human CSX/NKX-2.5 homeodomain and Drosophila NK-2 proteins have resolved long-standing questions on the role of protein stability and temperature dependence on DNA binding free energy.

使用分子建模和NMR光谱，三维结构的野生型和突变的同源结构域包含蛋白质的NK-2类，人类CSX/NKX-2.5，和NKX-3.1蛋白质，和独特的HOP同源结构域，已经研究了在游离状态下，结合到DNA，并在三元复合物与相关的转录因子。最新的结果集中在DNA特异性和增强子复合物组装的结构机制，包括人Nkx3.1，CSX/NKX-2.5和HOP与血清反应因子（SRF）的相互作用。这些同源异型蛋白的NMR揭示了新的相互作用基序和抑制机制，在心脏发生中起着至关重要的作用，随后通过NMR鉴定的区域中的突变的功能测定证实了这一点。对Nkx3.1（一种发现与前列腺癌风险共分离的致癌基因）的人类同源结构域T41 M突变体的研究表明，同源结构域是热不稳定的，尽管其结构和DNA结合在其他方面没有改变。比较人类CSX/NKX-2.5同源结构域和果蝇NK-2蛋白质的NMR和量热法研究解决了长期存在的关于蛋白质稳定性和温度依赖性对DNA结合自由能的作用的问题。