Oligomerization of the polycystin-2 C-terminal tail and effects on its Ca2+-binding properties.
Bottom Line: Consequently, trimerization does not further improve the affinity of Ca(2+) binding in the SUPC2 Ccore relative to the isolated EF-hand domain.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.
Affiliation: From the Departments of Laboratory Medicine, Pharmacology, and firstname.lastname@example.org.Show MeSH
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Mentions: To determine whether there are additional Ca2+-binding sites outside the EF-hand region, we needed to be certain that both the binding stoichiometry and the binding affinity of the HPC Cterm were measured with a high degree of accuracy. Because Ca2+ is necessary for the expression and purification of the protein, it is inherently difficult to maintain a Ca2+-free protein sample without the use of Ca2+ chelators. If the KD for the Ca2+-binding affinity of the protein lies in a low micromolar range, even a micromolar level of residual Ca2+ in the sample will render a significant proportion of protein unavailable for ITC characterization. To account for the residual Ca2+ bound to the protein, two ITC experiments were set up to pair each “Ca2+-HPC2Cterm” experiment (Fig. 3A) with a matching “Ca2+-HPC2Cterm + chelator” experiment (Fig. 3B). The thermodynamic parameters of the binding interaction were determined by fitting both binding isotherms simultaneously to an identical binding site model (Fig. 3C). The presence of the Ca2+ chelator enabled us for the first time to definitively determine the binding stoichiometry, N, by accounting for the effects of residual Ca2+. The best fit values for binding stoichiometry are very close to 1 (Table 2), indicating that there is only one Ca2+-binding site, presumed to be the same Ca2+-binding site previously identified in the HPC2 C-EF. The best fit value for the binding affinity KD is 22 μm. Compared with the known HPC2 C-EF binding affinity (Kd ∼461 μm) (17), the Ca2+-binding affinity is significantly enhanced (20-fold increase) in the longer and trimeric HPC2 Cterm construct.
Affiliation: From the Departments of Laboratory Medicine, Pharmacology, and email@example.com.