title: Oligomerization of the polycystin-2 C-terminal tail and effects on its Ca2+-binding properties.
abstract: Polycystin-2 (PC2) belongs to the transient receptor potential (TRP) family and forms a Ca(2+)-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 Ca(2+)-binding profiles in the C-terminal tail using biophysical approaches. Specifically, we determined that HPC2 Cterm forms a trimer in solution with and without Ca(2+) bound, although TRP channels are believed to be tetramers. We found that there is only one Ca(2+)-binding site in the HPC2 Cterm, located within its EF-hand domain. However, the Ca(2+) 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 Ca(2+) binding. In contrast to the human PC2, the SUPC2 Ccore contains two cooperative Ca(2+)-binding sites within its EF-hand domain. Consequently, trimerization does not further improve the affinity of Ca(2+) binding in the SUPC2 Ccore relative to the isolated EF-hand domain. Using NMR, we localized the Ca(2+)-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 Ca(2+)-dependent regulation of the PC2 channel by its cytosolic C-terminal domain. The improved methodology also serves as a good strategy to characterize other Ca(2+)-binding proteins.
authors.affiliation: From the Departments of Laboratory Medicine, Pharmacology, and yifei.yang@yale.edu.
authors.forename: Yifei
authors.initials: Y
authors.lastname: Yang
authors.affiliation: From the Departments of Laboratory Medicine.
authors.forename: Camille
authors.initials: C
authors.lastname: Keeler
authors.affiliation: Pharmacology, and.
authors.forename: Ivana Y
authors.initials: IY
authors.lastname: Kuo
authors.affiliation: Pharmacology, and.
authors.forename: Elias J
authors.initials: EJ
authors.lastname: Lolis
authors.affiliation: Pharmacology, and Cellular and Molecular Physiology, School of Medicine, Yale University, New Haven, Connecticut 06520.
authors.forename: Barbara E
authors.initials: BE
authors.lastname: Ehrlich
authors.affiliation: From the Departments of Laboratory Medicine, michael.hodsdon@yale.edu.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Hodsdon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating; D017136:Ion Transport; D008958:Models, Molecular; D008969:Molecular Sequence Data; D066329:Protein Aggregates; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012617:Sea Urchins / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D013045:Species Specificity; D050396:TRPP Cation Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D002118:Calcium; D066329:Protein Aggregates; D020033:Protein Isoforms; D011994:Recombinant Proteins; D050396:TRPP Cation Channels
keywords: Calcium; Calcium Intracellular Release; Calcium-binding Protein; EF-hand Proteins; Isothermal Titration Calorimetry (ITC); Nuclear Magnetic Resonance (NMR); Polycystic Kidney Disease