Molecular basis for polycystin-2 channel regulation and assembly via its C-terminal tail

Polycystin-2 (PC2) belongs to the transient receptor potential (TRP) family. PC2 can form a Cat2+-permeable channel in the membrane and it is important for intracellular Cat2+-signaling. The activity of PC2 channel can be regulated by changes in cytosolic Cat 2+-levels. Mutations in the C-terminal tail of human PC2 (HPC2 Cterm) can lead to autosomal dominant poly-cystic kidney disease (ADPKD). The HPC2 Cterm is also important for PC2 channel functions. The HPC2 Cterm contains the Cat2+-binding site necessary for the Cat2+-dependent activation of PC2 channel, and it also contains a coiled-coil domain that is involved in PC2 channel assembly and hetero-oligomerization with other proteins. The molecular mechanism linking mutations in PC2 and the pathogenesis of ADPKD is not well understood. Therefore, understanding the functional regulation of PC2 and its interaction with other proteins under both physiologic and pathogenic conditions is important for elucidating the disease mechanism and identifying potential molecular targets for treatment.;To provide the molecular basis for understanding how the C-terminal tail of PC2 contributes to the channel function and assembly, the presented thesis work is designed to characterize the oligomerization and Ca2+-binding within the PC2 C-terminal domain. I first determine the biophysical properties of the oligomerization and Ca2+-binding interaction of the PC2 Cterm. Furthermore, I use both solution nuclear magnetic resonance (NMR) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to characterize the structural and dynamic responses to oligomerization and Ca2+-binding within the PC2 C-terminal tail. In addition to human PC2 (HPC2), the sea urchin orthologue (SUPC2) is also investigated in parallel to understand the structure and function relationship between the number of Ca2+-binding sites and the overall Ca2+-dependent activity of the PC2 channel.;In Chapter 2, I describe the oligomeric states of different PC2 C-terminal constructs, in order to understand the effects of the coiled-coil region on the overall oligomerization properties of the PC2 C-terminal tail. The PC2 Cterm forms a trimer in solution, independent of Ca2+ presence. The trimer formation is mediated through the coiled-coil region. In Chapter 3, I develop and apply an alternative framework to characterize and analyze the Cat2+-binding properties of both human and sea urchin PC2 C-terminal domains using isothermal titration calorimetry (ITC). The ITC analysis indicates that the EF-hand domain is responsible for Cat2+-binding of the PC2 Cterm. The coiled-coil region, in comparison, is not required for Cat2+-binding. The binding profiles serve as the thermodynamic basis for defining the relationship between Cat2+-binding and structural stabilization within the PC2 Cterm. In Chapter 4 and 5, I identify the conformational and dynamic changes in response to Cat2+-binding within the PC2 Cterm. In both human and sea urchin PC2, Cat2+-binding stabilizes the EF-hand region, and alters its conformation. By comparing different PC2 C-terminal constructs, I also show that the coiled-coil domain and oligomerization can affect the molecular motions of the PC2 C-terminal tail.;By applying a combination of structural and biophysical approaches, I define the oligomerization and Cat2+-binding interaction within the PC2 Cterm. The study presents the first complete map of dynamic responses to Cat2+ within the PC2 C-terminal tail. The results not only provide the molecular evidence for the functional importance of PC2 C-terminal tail, but also suggest mechanisms for PC2 channel assembly and functional regulation through its C-terminal tail.