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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

Backbone chemical shift comparison of SUPC2 Ccore and SUPC2 C-EF in the EF-hand domain.A, averaged chemical shift change of each residue between SUPC2 C-EF and SUPC2 Ccore based on five sets of nuclei. Averaged chemical shift change for each residue is calculated as stated in Equation 1. Regions of secondary structure are indicated below with block arrows representing β bridges and cylinders representing helical regions. B, an overlay of the results from the backbone chemical shifts changes on the NMR structure of the SUPC2 C-EF. Greater chemical shift changes are displayed as increasing green intensity. Lower chemical shift changes are displayed as increasing blue intensity. Light gray indicates residues for which chemical shift data are not available. The figure at the right is rotated 180° around the y axis.
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Figure 5: Backbone chemical shift comparison of SUPC2 Ccore and SUPC2 C-EF in the EF-hand domain.A, averaged chemical shift change of each residue between SUPC2 C-EF and SUPC2 Ccore based on five sets of nuclei. Averaged chemical shift change for each residue is calculated as stated in Equation 1. Regions of secondary structure are indicated below with block arrows representing β bridges and cylinders representing helical regions. B, an overlay of the results from the backbone chemical shifts changes on the NMR structure of the SUPC2 C-EF. Greater chemical shift changes are displayed as increasing green intensity. Lower chemical shift changes are displayed as increasing blue intensity. Light gray indicates residues for which chemical shift data are not available. The figure at the right is rotated 180° around the y axis.

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.


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)

Backbone chemical shift comparison of SUPC2 Ccore and SUPC2 C-EF in the EF-hand domain.A, averaged chemical shift change of each residue between SUPC2 C-EF and SUPC2 Ccore based on five sets of nuclei. Averaged chemical shift change for each residue is calculated as stated in Equation 1. Regions of secondary structure are indicated below with block arrows representing β bridges and cylinders representing helical regions. B, an overlay of the results from the backbone chemical shifts changes on the NMR structure of the SUPC2 C-EF. Greater chemical shift changes are displayed as increasing green intensity. Lower chemical shift changes are displayed as increasing blue intensity. Light gray indicates residues for which chemical shift data are not available. The figure at the right is rotated 180° around the y axis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Backbone chemical shift comparison of SUPC2 Ccore and SUPC2 C-EF in the EF-hand domain.A, averaged chemical shift change of each residue between SUPC2 C-EF and SUPC2 Ccore based on five sets of nuclei. Averaged chemical shift change for each residue is calculated as stated in Equation 1. Regions of secondary structure are indicated below with block arrows representing β bridges and cylinders representing helical regions. B, an overlay of the results from the backbone chemical shifts changes on the NMR structure of the SUPC2 C-EF. Greater chemical shift changes are displayed as increasing green intensity. Lower chemical shift changes are displayed as increasing blue intensity. Light gray indicates residues for which chemical shift data are not available. The figure at the right is rotated 180° around the y axis.
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.

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