Yang Y et al. (2015), Oligomerization of the polycystin-2 C-terminal ...

ECB-ART-43849

J Biol Chem 2015 Apr 17;29016:10544-54. doi: 10.1074/jbc.M115.641803.

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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 .

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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.

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Genes referenced: LOC100887844 LOC100893907 LOC575928 LOC593358 pcsk2 pkd2 srpl

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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.
	FIGURE 2. SEC-MALS results of HPC2 Cterm and SUPC2 Ccore in Ca2+-bound holo and Ca2+-free apo states. A, three different amounts of HPC2 Cterm protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca2+-saturating buffer (20 mm CaCl2). The UV curves indicate the elution peaks of the protein samples analyzed, and the dotted lines indicate the calculated molar mass across the elution peaks. Orange, 1.16-mg injection; green, 155-μg injection; blue, 31.0-μg injection. B, three different amounts of HPC 2Cterm protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca2+-free buffer (no added CaCl2, 1 mm EDTA). Orange, 2.36-mg injection; green, 650-μg injection; blue, 260-μg injection. C, three different amounts of SUPC2 Ccore protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca2+-saturating buffer (20 mm CaCl2). The UV curves indicate the elution peaks of the protein samples analyzed, and the dotted lines indicate the calculated molar mass across the elution peaks. Orange, 1.35-mg injection; green, 150-μg injection; blue, 15.0-μg injection. D, three different amounts of SUPC2 Ccore protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca2+-free buffer (no added CaCl2, 1 mm EDTA). Orange, 1.35-mg injection; green, 150-μg injection; blue, 75.2 μg injection.
	FIGURE 3. ITC measurement of PC2 C-terminal constructs and Ca2+ binding interaction. A, raw heat measurement of 1.96 mm CaCl2 titrated into 100 μm HPC2 Cterm protein in pH 7.4, 25 mm Tris, 150 mm KCl, 20 mm imidazole buffer at 25 °C, using 32 injections of 1.49 μl/injection. B, raw heat measurement of 1.96 mm CaCl2 titrated into 98.5 μm HPC2 Cterm protein and 115 μm EDTA in pH 7.4, 25 mm Tris, 150 mm KCl, 20 mm imidazole buffer at 25 °C, using 32 injections of 1.49 μl/injection. C, simultaneously fitted, baseline-corrected isotherms of CaCl2 into HPC2 Cterm only (blue trace) and protein with EDTA (red trace) ITC experiment. D, raw heat measurement of 1.00 mm CaCl2 titrated to 15.2 μm SUPC2 C-EF protein in pH 7.4, 25 mm Tris, 150 mm KCl, 1 mm TCEP buffer at 25 °C, using 36 injections of 8 μl/injection. E, raw heat measurement of 1.00 mm CaCl2 titrated into 13.3 μm SUPC2 C-EF protein and 88 μm 5,5′-dimethyl-BAPTA in pH 7.4, 25 mm Tris, 150 mm KCl, 1 mm TCEP buffer at 25 °C, using 36 injections of 8 μl/injection. F, simultaneously fitted, baseline-corrected isotherms of SUPC2 C-EF only (blue trace) and SUPC2 C-EF with 5,5′-dimethyl-BAPTA (red trace) ITC experiment. G, raw heat measurement of 2.44 mm CaCl2 titrated into 88 μm SUPC2 Ccore protein in pH 7.4, 25 mm Tris, 150 mm KCl, 1 mm TCEP buffer at 25 °C, using 32 injections of 1.49 μl/injection. H, raw heat measurement of 2.44 mm CaCl2 titrated into 86 μm SUPC2 Ccore protein and 255 μm EDTA in pH 7.4, 25 mm Tris, 150 mm KCl, 1 mm TCEP buffer at 25 °C, using 32 injections of 1.49 μl/injection. I, simultaneously fitted, baseline-corrected isotherms of SUPC2 Ccore only (blue trace) and SUPC2 Ccore with EDTA (red trace) ITC experiment.
	FIGURE 4. NMR spectra of SUPC2 Ccore under holo and apo conditions. Comparison of 1H-15N HSQC NMR spectra of 13C15N SUPC2 Ccore in the Ca2+-saturating condition (red contours) and in the Ca2+-free condition (green contours) in pH 7.4, 2 mm Tris-d11, 150 mm KCl buffer with 1 mm TCEP at 25 °C.
	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.

References [+] :

Allen, A high-resolution structure of the EF-hand domain of human polycystin-2. 2014, Pubmed