SELENO-METHIONINE DERIVATIVE OF THE KH3 DOMAIN OF THE FUSE BINDING PROTEIN

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。FUSE Binding Protein （FBP）与c-myc癌基因远上游元件（FUSE）结合，导致c-myc转录增强。这种结合是由FBP的四个KH结构域介导的，每个结构域识别FUSE中的单链一致序列。了解KH结构域如何识别和区分DNA物种，可以深入了解myc激活及其过程，如细胞增殖和肿瘤发生。FBP的第三和第四个KH结构域（KH3和KH4）的核磁共振结构已经确定，每个结构域都与同源的ssDNA序列结合（Braddock等人，Nature 415, pgs）。1051 - 1056, 2002);然而，到目前为止还没有确定晶体结构。这项工作是正在进行的设计抑制DNA与FBP结合的小分子的一部分。c-myc蛋白对细胞增殖很重要，但必须适当调节以防止过度生长和肿瘤形成。由于fbp与FUSE的结合促进了c-myc的转录，在c-myc过度活跃的情况下，这种相互作用的破坏可能导致肿瘤形成的阻止或逆转。这种结构还可以深入了解分子识别，而分子识别是这些重要生物过程的核心。它还可以揭示水在这种相互作用中所起的作用，这在核磁共振结构中是看不到的。我们培养了与selex优化的七聚体ssDNA结合的KH3结构域晶体，并在NSLS的X29站收集了数据。数据集处理至2.6埃；然而，试图通过分子替代来解决该结构失败了。我们建议收集与同一七聚体DNA序列结合的硒-蛋氨酸KH3结构域晶体的数据，以便使用硒MAD来解决该结构。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The FUSE Binding Protein (FBP) binds to the far upstream element (FUSE) of the c-myc oncogene, resulting in enhancement of c-myc transcription. This binding is mediated by four KH domains of FBP, each of which recognizes a single stranded consensus sequence within the FUSE. Understanding how KH domains recognize and discriminate amongst DNA species can provide insight into myc activation and processes resulting from it, such as cellular proliferation and tumorigenesis. NMR structures of the third and fourth KH domains (KH3 and KH4) of FBP, each bound to a cognate ssDNA sequence have been determined (Braddock et. al. Nature 415, pgs. 1051-1056, 2002); however, no crystallographic structure has been determined thus far. This work is part of ongoing efforts to design small molecules that inhibit DNA binding to FBP. The c-myc protein is important for cellular proliferation, but must be properly regulated to prevent excess growth and tumor formation. Because FBP-binding to the FUSE promotes c-myc transcription, disruption of this interaction in cases of c-myc over-activity may result in arresting, or the reversal of, tumor formation. This structure can also yield insight into the molecular recognition that is at the core of these important biological processes. It can also reveal the role played by water, which is not seen in the NMR structures, in this interaction. We have grown crystals of the KH3 domain bound to a SELEX-optimzed heptameric ssDNA and collected data at station X29 of the NSLS. A data set was processed to 2.6 Angstroms; however, attempts to solve the structure by molecular replacement failed. We propose to collect data from crystals of seleno-methionine KH3 domain bound to the same heptameric DNA sequence in order to solve the structure using selenium MAD.