DARTMOUTH COL COBRE: P3: STRUCTURAL ANALYSIS OF CFTR PROTEINS INTERACTION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 囊性纤维化跨膜传导调节因子（CFTR）是一种上皮氯离子通道，在电解质和体液稳态中起关键作用。CFTR基因的突变会导致囊性纤维化，而肠道中细菌肠毒素对该通道的过度刺激会导致分泌性腹泻。因此，了解调节CFTR通道活性的机制可能对控制这些疾病很重要。已经积累的证据表明，不同的结合相互作用控制CFTR的成熟及其向质膜的转运。不同的结合伴侣介导不同的运输效应，并且CFTR表达的总体水平可能由它们的竞争影响的相互作用决定。我们采取了双管齐下的方法来研究这些监管相互作用的结构基础。首先，我们将研究一系列将重组表达的CFTR结合PDZ结构域的结合参数。结合动力学和热力学正在使用表面等离子体共振和荧光偏振各向异性技术进行测定，并将用于评估不同结构域竞争CFTR结合的能力。其次，通过同源建模预测了CAL PDZ结构域的立体化学，并用于指导靶向突变以研究PDZ相互作用的功能作用。同时，该结构域和其他CFTR结合PDZ结构域的结构将通过与Mierke实验室（布朗大学）合作的NMR来确定。最终，这些结构和功能的见解可以为设计化合物提供基础，这些化合物可以通过靶向稳定或破坏调节CFTR功能的特定蛋白质-蛋白质相互作用来影响疾病进展。

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 cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial chloride channel that plays a key role in electrolyte and fluid homeostasis. Mutations in the CFTR gene cause cystic fibrosis, whereas overstimulation of the channel by bacterial enterotoxins in the gut leads to secretory diarrhea. Thus, understanding the mechanisms that regulate CFTR channel activity may be important in controlling these diseases. Evidence has accumulated that diverse binding interactions control both the maturation of CFTR and its transport to the plasma membrane. Different binding partners mediate different trafficking effects, and it is likely that the overall level of CFTR expression is determined by the interplay of their competing influences. We have taken a two-pronged approach to investigate the structural basis of these regulatory interactions. First, we will investigate the binding parameters of a series of CFTR-binding PDZ domains that will be recombinantly expressed. Binding kinetics and thermodynamics are being determined using surface plasmon resonance and fluorescence polarization anisotropy techniques, and will be used to assess the ability of different domains to compete for CFTR binding. Secondly, the stereochemistry of the CAL PDZ domain has been predicted by homology modeling, and used to guide targeted mutagenesis to investigate the functional role of the PDZ interaction. In parallel, the structure of the domain and other CFTR-binding PDZ domains will be determined by NMR in collaboration with the Mierke lab (Brown University). Ultimately, these structural and functional insights may provide the basis for designing compounds that can influence disease progression by targeted stabilization or disruption of particular protein-protein interactions that regulate CFTR function.