TY - JOUR
T1 - Oligomerization of the polycystin-2 C-terminal tail and effects on its Ca2+-binding properties
AU - Yang, Yifei
AU - Keeler, Camille
AU - Kuo, Ivana Y.
AU - Lolis, Elias J.
AU - Ehrlich, Barbara E.
AU - Hodsdon, Michael E.
N1 - Publisher Copyright:
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2015/4/17
Y1 - 2015/4/17
N2 - 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, although 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.
AB - 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, although 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.
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U2 - 10.1074/jbc.M115.641803
DO - 10.1074/jbc.M115.641803
M3 - Article
C2 - 25716316
AN - SCOPUS:84927723607
SN - 0021-9258
VL - 290
SP - 10544
EP - 10554
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 16
ER -