Three-dimensional Structures Of Biological Macromolecule

利用分子模型和核磁共振波谱技术，研究了NK-2类、人类CSX/NKX-2.5和NKX-3.1蛋白的野生型和突变型同源结构域，以及独特的HOP同源结构域在自由状态、与DNA结合以及与相关转录因子三元配合物中的三维结构。最近的研究结果主要集中在DNA特异性和增强子复合物组装的结构机制上，包括人类Nkx3.1、CSX/NKX-2.5和HOP与血清反应因子（SRF）的相互作用。这些同型蛋白的核磁共振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，随后通过核磁共振鉴定的区域突变的功能分析证实了这一点。对发现与前列腺癌风险共分离的致癌基因Nkx3.1的人类同位结构域T41M突变体的研究表明，尽管其结构和DNA结合没有改变，但该同位结构域是热不稳定的。比较人类CSX/NKX-2.5同位结构域和果蝇NK-2蛋白的核磁共振和量热研究解决了长期以来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。

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.