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

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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.