Oligomerization of the polycystin-2 C-terminal tail and the effects on its Ca2+-binding properties.

Polycystin-2 (PC2) belongs to the transient receptor potential (TRP) family and forms a Ca2+-regulated channel. The C-terminal cytoplasmic tail of human PC2 (HPC2 Cterm) is important for PC2 channel assembly and regulation. In this study, we characterized the oligomeric states and Ca2+-binding profiles in the C-terminal tail using biophysical approaches. Specifically, we determined that HPC2 Cterm forms a trimer in solution with and without Ca2+ bound, even though TRP channels are believed to be tetramers. We found that there is only one Ca2+-binding site in the HPC2 Cterm, located within its EF-hand domain. However, the Ca2+ binding affinity of the HPC2 Cterm trimer is greatly enhanced relative to the intrinsic binding affinity of the isolated EF-hand domain. We also employed the sea urchin PC2 (SUPC2) as a model for biophysical and structural characterization. The sea urchin C-terminal construct (SUPC2 Ccore) also forms trimers in solution, independent of Ca2+ binding. In contrast to the human PC2, the SUPC2 Ccore contains two cooperative Ca2+-binding sites within its EF-hand domain. Consequently, trimerization does not further improve the affinity of Ca2+ binding in the SUPC2 Ccore relative to the isolated EF-hand domain. Using NMR, we localized the Ca2+-binding sites in the SUPC2 Ccore and characterized the conformational changes in its EF-hand domain due to trimer formation. Our study provides a structural basis for understanding the Ca2+-dependent regulation of the PC2 channel by its cytosolic C-terminal domain. The improved methodology also serves as a good strategy to characterize other Ca2+-binding proteins.