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

Yang Y, Keeler C, Kuo IY, Lolis EJ, Ehrlich BE, Hodsdon ME - J. Biol. Chem. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: From the Departments of Laboratory Medicine, Pharmacology, and yifei.yang@yale.edu.

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Related in: MedlinePlus

The PC2 C-terminal domains in human and sea urchin PC2.A, alignment of the C-terminal domain sequence of human and sea urchin PC2 homologues. B, different domains are included in the construct design. The constructs are positioned based on sequence alignment results. The residue numbers mark the start and finish residues of each construct, in the context of full-length human and sea urchin PC2 proteins, respectively. Therefore, the human and sea urchin constructs are numbered differently.
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Figure 1: The PC2 C-terminal domains in human and sea urchin PC2.A, alignment of the C-terminal domain sequence of human and sea urchin PC2 homologues. B, different domains are included in the construct design. The constructs are positioned based on sequence alignment results. The residue numbers mark the start and finish residues of each construct, in the context of full-length human and sea urchin PC2 proteins, respectively. Therefore, the human and sea urchin constructs are numbered differently.

Mentions: Characterizing the Ca2+-binding elements postulated to reside within the PC2 C-terminal tail is crucial for understanding the mechanism of channel regulation. Based on the bell-shaped response of open probability to increasing Ca2+ concentration, it can be hypothesized that separate Ca2+-binding sites are responsible for different sides of the bell-shaped curve (4, 5, 7, 9, 16). The EF-hand domain (amino acids 719–798 of human PC2) contains one Ca2+-binding site, which is important for the Ca2+-dependent regulation of PC2 channel (7, 9). However, the isolated EF-hand domain binds Ca2+ very weakly, with a KD of 461 μm, outside the physiologic range of cytosolic Ca2+ concentrations (17). Therefore, it was speculated that there could be additional Ca2+-response elements located in the C-terminal domain outside the EF-hand region (7). Moreover, a series of acidic residues located in the loop region (linker 2-L2) (Fig. 1A) connecting the EF-hand and coiled-coil domain share a sequence similar to the Ca2+-bowl structure found in the BK (big potassium) channel (18, 19). It was important to determine the Ca2+-binding profiles of HPC2 Cterm because this would address the question of whether there are additional C-terminal Ca2+-binding sites outside the EF-hand domain and if the Ca2+-binding affinity of HPC2 Cterm is within the physiologic range (nanomolar to low micromolar). However, the Ca2+-binding profile of the HPC2 Cterm was not fully determined in previous studies using isothermal titration calorimetry (ITC) due to the presence of residual Ca2+ and protein aggregates (7).


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

Yang Y, Keeler C, Kuo IY, Lolis EJ, Ehrlich BE, Hodsdon ME - J. Biol. Chem. (2015)

The PC2 C-terminal domains in human and sea urchin PC2.A, alignment of the C-terminal domain sequence of human and sea urchin PC2 homologues. B, different domains are included in the construct design. The constructs are positioned based on sequence alignment results. The residue numbers mark the start and finish residues of each construct, in the context of full-length human and sea urchin PC2 proteins, respectively. Therefore, the human and sea urchin constructs are numbered differently.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400361&req=5

Figure 1: The PC2 C-terminal domains in human and sea urchin PC2.A, alignment of the C-terminal domain sequence of human and sea urchin PC2 homologues. B, different domains are included in the construct design. The constructs are positioned based on sequence alignment results. The residue numbers mark the start and finish residues of each construct, in the context of full-length human and sea urchin PC2 proteins, respectively. Therefore, the human and sea urchin constructs are numbered differently.
Mentions: Characterizing the Ca2+-binding elements postulated to reside within the PC2 C-terminal tail is crucial for understanding the mechanism of channel regulation. Based on the bell-shaped response of open probability to increasing Ca2+ concentration, it can be hypothesized that separate Ca2+-binding sites are responsible for different sides of the bell-shaped curve (4, 5, 7, 9, 16). The EF-hand domain (amino acids 719–798 of human PC2) contains one Ca2+-binding site, which is important for the Ca2+-dependent regulation of PC2 channel (7, 9). However, the isolated EF-hand domain binds Ca2+ very weakly, with a KD of 461 μm, outside the physiologic range of cytosolic Ca2+ concentrations (17). Therefore, it was speculated that there could be additional Ca2+-response elements located in the C-terminal domain outside the EF-hand region (7). Moreover, a series of acidic residues located in the loop region (linker 2-L2) (Fig. 1A) connecting the EF-hand and coiled-coil domain share a sequence similar to the Ca2+-bowl structure found in the BK (big potassium) channel (18, 19). It was important to determine the Ca2+-binding profiles of HPC2 Cterm because this would address the question of whether there are additional C-terminal Ca2+-binding sites outside the EF-hand domain and if the Ca2+-binding affinity of HPC2 Cterm is within the physiologic range (nanomolar to low micromolar). However, the Ca2+-binding profile of the HPC2 Cterm was not fully determined in previous studies using isothermal titration calorimetry (ITC) due to the presence of residual Ca2+ and protein aggregates (7).

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.

View Article: PubMed Central - PubMed

Affiliation: From the Departments of Laboratory Medicine, Pharmacology, and yifei.yang@yale.edu.

Show MeSH
Related in: MedlinePlus