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 email@example.com.Show MeSH
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Mentions: We mapped the chemical shift perturbations to determine whether there are any domain-domain interactions within the trimeric SUPC2 Ccore construct. We calculated the perturbations by comparing the chemical shifts of assigned backbone atoms in the SUPC2 C-EF and the SUPC2 Ccore under holo conditions. We found that the majority of the chemical shifts in the EF-hand domain remain unchanged in the longer and trimeric SUPC2 Ccore protein. Most importantly, the two paired α helix-loop-α helix structural motifs that form the EF-hand region remain undisrupted (Fig. 5). The largest changes in chemical shifts, still less than 0.2 ppm, were detected near the N and C termini of the EF-hand domain. These changes are consistent with a decreased helicity in these residues in the SUPC2 Ccore construct compared with the SUPC2 C-EF (36), most likely due to the designed mutations at the end of the helices to achieve protease resistance. The perturbed residues are involved in the interaction of helices α1 and α4. Such effects could explain the slightly lower Ca2+-binding affinity of the SUPC2 Ccore (Tables 3 and 4). However, because the majority of the EF-hand domain remains the same, the ITC results also indicate that the two protein constructs share similar Ca2+-binding profiles. Hence, we report that there are few structural differences between the EF-hand regions of the two constructs. Our results suggest that there are no interactions between the EF-hand and coiled-coil/L2 linker in the SU Ccore protein or among different EF-hand domains in the SUPC2 Ccore trimer complex.
Affiliation: From the Departments of Laboratory Medicine, Pharmacology, and firstname.lastname@example.org.