Domain Mapping of the Polycystin-2 C-terminal Tail Using de Novo Molecular Modeling and Biophysical Analysis

In polycystic kidney disease (PKD), polycystin-2 (PC2) is frequently mutated or truncated in the C-terminal cytoplasmic tail (PC2-C). The currently accepted model of PC2-C consists of an EF-hand motif overlapping with a short coiled coil; however, this model fails to explain the mechanisms by which PC2 truncations C-terminal to this region lead to PKD. Moreover, direct PC2 binding to inositol 1,4,5-trisphosphate receptor, KIF3A, and TRPC1 requires residues in PC2-C outside this region. To address these discrepancies and investigate the role of PC2-C in PC2 function, we performed de novo molecular modeling and biophysical analysis. De novo molecular modeling of PC2-C using the ROBETTA server predicts two domains as follows: an EF-hand motif (PC2-EF) connected by a linker to a previously unidentified C-terminal coiled coil (PC2-CC). This model differs substantially from the current model and correlates with limited proteolysis, matrix-assisted laser desorption/ionization mass spectroscopy, N-terminal sequencing, and improved coiled coil prediction algorithms. PC2-C is elongated and oligomerizes through PC2-CC, as measured by analytical ultracentrifugation and size exclusion chromatography, whereas PC2-EF is globular and monomeric. We show that PC2-C and PC2-EF have micromolar affinity for calcium (Ca2+) by isothermal titration calorimetry and undergo Ca2+-induced conformational changes by circular dichroism. Mutation of predicted EF-hand loop residues in PC2 to alanine abolishes Ca2+ binding. Our results suggest that PC2-CC is involved in PC2 oligomerization, and PC2-EF is a Ca2+-sensitive switch. PKD-associated PC2 mutations are located in regions that may disrupt these functions, providing structural insight into how PC2 mutations lead to disease. In polycystic kidney disease (PKD), polycystin-2 (PC2) is frequently mutated or truncated in the C-terminal cytoplasmic tail (PC2-C). The currently accepted model of PC2-C consists of an EF-hand motif overlapping with a short coiled coil; however, this model fails to explain the mechanisms by which PC2 truncations C-terminal to this region lead to PKD. Moreover, direct PC2 binding to inositol 1,4,5-trisphosphate receptor, KIF3A, and TRPC1 requires residues in PC2-C outside this region. To address these discrepancies and investigate the role of PC2-C in PC2 function, we performed de novo molecular modeling and biophysical analysis. De novo molecular modeling of PC2-C using the ROBETTA server predicts two domains as follows: an EF-hand motif (PC2-EF) connected by a linker to a previously unidentified C-terminal coiled coil (PC2-CC). This model differs substantially from the current model and correlates with limited proteolysis, matrix-assisted laser desorption/ionization mass spectroscopy, N-terminal sequencing, and improved coiled coil prediction algorithms. PC2-C is elongated and oligomerizes through PC2-CC, as measured by analytical ultracentrifugation and size exclusion chromatography, whereas PC2-EF is globular and monomeric. We show that PC2-C and PC2-EF have micromolar affinity for calcium (Ca2+) by isothermal titration calorimetry and undergo Ca2+-induced conformational changes by circular dichroism. Mutation of predicted EF-hand loop residues in PC2 to alanine abolishes Ca2+ binding. Our results suggest that PC2-CC is involved in PC2 oligomerization, and PC2-EF is a Ca2+-sensitive switch. PKD-associated PC2 mutations are located in regions that may disrupt these functions, providing structural insight into how PC2 mutations lead to disease. Polycystic kidney disease (PKD) 4The abbreviations used are: PKD, polycystic kidney disease; PC2, polycystin-2; PC2-C, polycystin-2 C-terminal cytoplasmic domain; PC2-EF, polycystin-2 EF-hand domain; PC2-CC, polycystin-2 coiled coil domain; ITC, isothermal titration calorimetry; AUC, analytical ultracentrifugation; MALDI-MS, matrix-assisted laser desorption/ionization mass spectroscopy; 2DSA, two-dimensional spectrum analysis; SEC, size exclusion chromatography. is among the most common life-threatening inherited disorders, with clinical consequences characterized by renal and hepatic cysts (1Wu G. D'Agati V. Cai Y. Markowitz G. Park J.H. Reynolds D.M. Maeda Y. Le T.C. Hou Jr., H. Kucherlapati R. Edelmann W. Somlo S. Cell. 1998; 93: 7-188Google Scholar). Most cases of PKD (>95%) are linked with mutations in the genes Pkd1 or Pkd2, which encode the membrane protein polycystin-1 (PC1) and the calcium (Ca2+)-permeable channel, polycystin-2 (PC2), respectively (2Somlo S. Ehrlich B. Curr. Biol. 2001; 11: R356-360Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 3Clapham D.E. Nature. 2003; 426: 517-524Crossref PubMed Scopus (2199) Google Scholar). PC2 belongs to the TRP channel family and is expressed in most tissues (4Gonzalez-Perrett S. Kim K. Ibarra C. Damiano A.E. Zotta E. Batelli M. Harris P.C. Reisin I.L. Arnaout M.A. Cantiello H.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1182-1187Crossref PubMed Scopus (404) Google Scholar, 5Koulen P. Cai Y. Geng L. Maeda Y. Nishimura S. Witzgall R. Ehrlich B.E. Somlo S. Nat. Cell Biol. 2002; 4: 191-197Crossref PubMed Scopus (578) Google Scholar, 6Mochizuki T. Wu G. Hayashi T. Xenophontos S.L. Veldhuisen B. Saris J.J. Reynolds D.M. Cai Y. Gabow P.A. Pierides A. Kimberling W.J. Breuning M.H. Deltas C.C. Peters D.J. Somlo S. Science. 1996; 272: 1339-1342Crossref PubMed Scopus (1216) Google Scholar, 7Anyatonwu G.I. Ehrlich B.E. J. Biol. Chem. 2005; 280: 29488-29493Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 8Luo Y. Vassilev P.M. Li X. Kawanabe Y. Zhou J. Mol. Cell. Biol. 2003; 23: 2600-2607Crossref PubMed Scopus (158) Google Scholar). It has six transmembrane spans, and both C and N termini are cytoplasmic. PC1 and PC2 interact directly and co-localize to primary cilia where they are hypothesized to be necessary for a mechanosensory or chemosensory response that triggers a rise in intracellular Ca2+ (9Somlo S. Markowitz G.S. Curr. Opin. Nephrol. Hypertens. 2000; 9: 385-394Crossref PubMed Scopus (27) Google Scholar). Many aspects of PC2 function are mediated by the cytoplasmic C-terminal tail (PC2-C), including co-assembly with PC1 through the PC1 C-terminal cytoplasmic tail (10Tsiokas L. Kim E. Arnould T. Sukhatme V.P. Walz G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6965-6970Crossref PubMed Scopus (424) Google Scholar, 11Xu G.M. Arnaout M.A. Genomics. 2002; 79: 87-94Crossref PubMed Scopus (23) Google Scholar, 12Qian F. Germino F.J. Cai Y. Zhang X. Somlo S. Germino G.G. Nat. Genet. 1997; 16: 179-183Crossref PubMed Scopus (568) Google Scholar, 13Hanaoka K. Qian F. Boletta A. Bhunia A.K. Piontek K. Tsiokas L. Sukhatme V.P. Guggino W.B. Germino G.G. Nature. 2000; 408: 990-994Crossref PubMed Scopus (681) Google Scholar). Two-thirds of pathogenic mutations in PC1 and >90% of pathogenic mutations in PC2 result in truncations of their C-terminal cytoplasmic regions and are predicted to abrogate interaction between the proteins. There are no structural data describing PC2, and current descriptions of PC2-C lack functional and biochemical information. The currently accepted domain model of PC2-C consists of an EF-hand motif overlapping with a short coiled coil (6Mochizuki T. Wu G. Hayashi T. Xenophontos S.L. Veldhuisen B. Saris J.J. Reynolds D.M. Cai Y. Gabow P.A. Pierides A. Kimberling W.J. Breuning M.H. Deltas C.C. Peters D.J. Somlo S. Science. 1996; 272: 1339-1342Crossref PubMed Scopus (1216) Google Scholar, 12Qian F. Germino F.J. Cai Y. Zhang X. Somlo S. Germino G.G. Nat. Genet. 1997; 16: 179-183Crossref PubMed Scopus (568) Google Scholar). Helical wheel projection of this purported coiled coil (Glu772–Leu796) shows that residues in positions “a” and “d” of the helix (i.e. the coiled coil interface) are hydrophilic or charged and would be unfavorable in a coiled coil interface (Fig. 1D). PKD-associated truncations have been identified outside of these proposed domains in PC2-C (Fig. 1F), suggesting that residues important for PC2 function are located in the truncated regions. Moreover, PC2 binds directly to several proteins, including the inositol 1,4,5-trisphosphate receptor (14Li Y. Wright J.M. Qian F. Germino G.G. Guggino W.B. J. Biol. Chem. 2005; 280: 41298-41306Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), TRPC1 (15Tsiokas L. Arnould T. Zhu C. Kim E. Walz G. Sukhatme V.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3934-3939Crossref PubMed Scopus (274) Google Scholar), tropomyosin-1 (16Li Q. Dai Y. Guo L. Liu Y. Hao C. Wu G. Basora N. Michalak M. Chen X.Z. J. Mol. Biol. 2003; 325: 949-962Crossref PubMed Scopus (73) Google Scholar), Id2 (17Li X. Luo Y. Starremans P.G. McNamara C.A. Pei Y. Zhou J. Nat. Cell Biol. 2005; 7: 1202-1212Crossref PubMed Scopus (171) Google Scholar), troponin-1 (18Li Q. Shen P.Y. Wu G. Chen X.Z. Biochemistry. 2003; 42: 450-457Crossref PubMed Scopus (65) Google Scholar), KIF3A (19Li Q. Montalbetti N. Wu Y. Ramos A. Raychowdhury M.K. Chen X.Z. Cantiello H.F. J. Biol. Chem. 2006; 281: 37566-37575Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), and PC1 (9Somlo S. Markowitz G.S. Curr. Opin. Nephrol. Hypertens. 2000; 9: 385-394Crossref PubMed Scopus (27) Google Scholar, 20Delmas P. Biol. Res. 2004; 37: 681-691Crossref PubMed Scopus (11) Google Scholar). All of these interactions are dependent on residues C-terminal to the currently accepted domain organization of PC2-C. To address these discrepancies and to aid future studies into the molecular basis of PKD pathogenesis and progression, we constructed a validated structural model of PC2-C. We have determined a de novo molecular model of PC2-C using the ROBETTA server (21Chivian D. Kim D.E. Malmstrom L. Schonbrun J. Rohl C.A. Baker D. Proteins. 2005; 61: 157-166Crossref PubMed Scopus (124) Google Scholar, 22Chivian D. Kim D.E. Malmstrom L. Bradley P. Robertson T. Murphy P. Strauss C.E. Bonneau R. Rohl C.A. Baker D. Proteins. 2003; 53: 524-533Crossref PubMed Scopus (253) Google Scholar, 23Kim D.E. Chivian D. Baker D. Nucleic Acids Res. 2004; 32: W526-W531Crossref PubMed Scopus (1442) Google Scholar) and validated characteristics of this model using biophysical and biochemical analysis. We show that PC2-C contains two domains, a single EF-hand motif (PC2-EF) connected by a linker to a coiled coil domain (PC2-CC). We propose that PC2-CC is the actual coiled coil domain that has been assumed in the literature. Our results suggest that PC2-CC is involved in PC2 oligomerization and that PC2-EF acts as a Ca2+-sensitive switch. PKD-associated PC2 truncation mutations are located in regions that would disrupt these functions, providing insight into how PC2 mutations may lead to disease. Protein Expression and Purification—Fragments of polycystin-2 are as follows. PC2-C (Ile704–Val968), PC2-EF (Asn720–Pro797), and PC2-CC (Gly828–His927) were PCR-amplified from human PC2 cDNA (obtained from S. Somlo, Yale University), cloned into pET-28 (a+) (Novagen), and transformed into BL21(DE3) CodonPlus RIL (Stratagene) for bacterial expression. Mutant variants of PC2-EF (PC2-EF-X-Z T771A/E774A) and PC2-CC (L842P, V846E, M849K, I853P, I856K, V846E/I856K, and M849K/V863E) were created using QuikChange multiple site-directed mutagenesis kit (Stratagene). Cells were grown to OD595 ∼0.4 at 37 °C and then shifted to 18 °C after induction with 1 mm isopropyl 1-thio-β-d-galactopyranoside at ∼18 h. Cells were harvested and resuspended in Buffer A (20 mm Tris, 500 mm NaCl, pH 8.0), lysed by freeze thaw/sonication with lysozyme (∼1 mg/ml) (Sigma), and clarified by centrifugation. Supernatant was loaded onto a 1-ml HisTrap column (GE Healthcare). The column was washed with 30 column volumes of Buffer A and then with 10 column volumes of Buffer A + 50 mm imidazole. Purified polycystin-2 fragments were eluted in Buffer A + 500 mm imidazole and applied to a Superdex 200 SEC column equilibrated in Buffer A. PC2-CC was treated with thrombin (1 unit/mg) for all experiments to remove the His tag. PC2-C and PC2-EF contain an N-terminal His tag with the sequence “MGSSHHHHHHSSGLVPRGSHM.” Molecular Modeling—PC2-C (Ile704–Val968) and PC2-EF (Asn720–Pro797) were submitted to the full-chain structure prediction server ROBETTA (21Chivian D. Kim D.E. Malmstrom L. Schonbrun J. Rohl C.A. Baker D. Proteins. 2005; 61: 157-166Crossref PubMed Scopus (124) Google Scholar, 22Chivian D. Kim D.E. Malmstrom L. Bradley P. Robertson T. Murphy P. Strauss C.E. Bonneau R. Rohl C.A. Baker D. Proteins. 2003; 53: 524-533Crossref PubMed Scopus (253) Google Scholar, 23Kim D.E. Chivian D. Baker D. Nucleic Acids Res. 2004; 32: W526-W531Crossref PubMed Scopus (1442) Google Scholar). Models were analyzed for structure using H. Nucleic Acids Res. 2004; 32: PubMed Scopus Google Scholar) and using for the ROBETTA are as The ROBETTA model for and were to a model of PC2-C for this model are in the data was treated with or in mm Tris, mm NaCl, pH were using were on to and for N-terminal were for results were analyzed using A. A. E. 2002; PubMed Scopus Google Scholar). were on an at °C with and PC2-C and PC2-EF were of Ca2+ with mm Protein were measured by (PC2-C), and and were in and at (PC2-C), and (PC2-CC). PC2-C mg/ml) and PC2-EF mg/ml) were in the of mm were were to using in were by data structure were performed using and N. 2000; PubMed Scopus Google Scholar, L. Nucleic Acids Res. 2004; 32: PubMed Scopus Google Scholar, A. L. 2002; PubMed Scopus Google Scholar). PC2-C and PC2-EF in the and of Ca2+ were with whereas PC2-CC were with N. 2000; PubMed Scopus Google Scholar). structure were with the structure of de novo of PC2-C (Ile704–Val968), PC2-EF or PC2-CC were on a PC2-C was in the and Ca2+ was in the PC2-EF and Ca2+ was were of Ca2+ with mm and in mm Tris, mm NaCl, pH of were at were to a model using and and of binding PC2-EF, or PC2-CC was applied to a Superdex 200 analytical SEC column (GE equilibrated in mm Tris, mm NaCl, pH at The column was using the and The was measured and where column was for PC2-C PC2-C with mm PC2-EF, and experiments were performed with a at the for of of of experiments were analyzed with B. D.J. and of Scholar). were S.L. in and Science. of Scholar), as in The volumes of PC2-EF, PC2-CC, and PC2-C were to and and as and Google Scholar), as in and data were to multiple and the on and of the and were on a at the of of and on of were analyzed in a mm experiments were performed at °C and were in in the of at for PC2-CC, for PC2-EF, and for PC2-C were at with size and no to for PC2-CC for PC2-EF and for PC2-C were to and PubMed Scopus Google Scholar). data were analyzed with E. B. of the on for Scholar) with B. E. Sci. Scopus Google Scholar), direct of of the B. H. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) with the as in B. 2004; PubMed Scopus Google Scholar). Molecular of the C-terminal of by a novo structural of PC2-C (Ile704–Val968) and PC2-EF were as from the ROBETTA server are as The PC2-C model predicts an elongated structure connected by a linker a PC2 Y. G. D. T. Qian Q. Geng L. R. Ehrlich B.E. Somlo S. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). 1 consists of a globular whereas domain contains a of coiled (Fig. domain 1 was as an EF-hand motif in the structure as from the ROBETTA residues to in Ca2+ binding were for Ca2+ in this and the de novo in ROBETTA is for single domain proteins, we submitted the sequence for the predicted EF-hand domain for de novo modeling to the ROBETTA the PC2-EF in a single EF-hand motif with a (Fig. The ROBETTA to or model the of a Ca2+ loop in model is in of the of a EF-hand motif in PC2-C. a structural domain with to a coiled coil was C-terminal to the of the purported coiled coil domain in the currently accepted model of PC2-C. To investigate the that a previously coiled coil motif is in the C-terminal cytoplasmic tail of PC2, the sequence of PC2-C was analyzed for coiled coil using the M. T. 2002; PubMed Scopus Google Scholar) which contains an prediction The from predicts that residues have a to a coiled coil residues were and to to a on the in the PC2-CC model (Fig. and and residues are and the coiled coil interface M. Protein Chem. 2005; PubMed Scopus Google Scholar). The results of de novo modeling for the cytoplasmic tail of PC2 suggest that PC2-CC is the actual coiled coil domain assumed in the literature. PC2-CC are necessary for direct binding to and and oligomerization (9Somlo S. Markowitz G.S. Curr. Opin. Nephrol. Hypertens. 2000; 9: 385-394Crossref PubMed Scopus (27) Google Scholar, Y. Wright J.M. Qian F. Germino G.G. Guggino W.B. J. Biol. Chem. 2005; 280: 41298-41306Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, L. Arnould T. Zhu C. Kim E. Walz G. Sukhatme V.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3934-3939Crossref PubMed Scopus (274) Google Scholar, Q. Dai Y. Guo L. Liu Y. Hao C. Wu G. Basora N. Michalak M. Chen X.Z. J. Mol. Biol. 2003; 325: 949-962Crossref PubMed Scopus (73) Google Scholar, X. Luo Y. Starremans P.G. McNamara C.A. Pei Y. Zhou J. Nat. Cell Biol. 2005; 7: 1202-1212Crossref PubMed Scopus (171) Google Scholar, Q. Shen P.Y. Wu G. Chen X.Z. Biochemistry. 2003; 42: 450-457Crossref PubMed Scopus (65) Google Scholar, Q. Montalbetti N. Wu Y. Ramos A. Raychowdhury M.K. Chen X.Z. Cantiello H.F. J. Biol. Chem. 2006; 281: 37566-37575Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 20Delmas P. Biol. Res. 2004; 37: 681-691Crossref PubMed Scopus (11) Google Scholar). PC2-CC the most C-terminal pathogenic PKD-associated truncation D.M. Hayashi T. Cai Y. Veldhuisen B. T. Qian F. Maeda Y. Li L. R. E. Wu G. Breuning M.H. Germino G.G. Peters D.J. Somlo S. J. Nephrol. 1999; PubMed Google Scholar). This truncation is to interact with the C of PC1 F. Germino F.J. Cai Y. Zhang X. Somlo S. Germino G.G. Nat. Genet. 1997; 16: 179-183Crossref PubMed Scopus (568) Google Scholar), to a for PKD modeling studies a previously coiled coil domain the C-terminal cytoplasmic tail of PC2, which may as an oligomerization interface by PKD-associated the and of the C-terminal of the of de novo structural we the domain structure of PC2-C using limited experiments with and N-terminal PC2-C (Ile704–Val968) was treated with or to fragments single (Fig. from were analyzed by N-terminal and to fragments that with the and from and and from both the predicted linker region of PC2-C the of these results the of an linker between residues and were analyzed by and with fragments of the PC2-C sequence using the N-terminal results as a to and results were modeling was structure prediction using the ROBETTA server several The residues to these PC2-C fragments were onto de novo model of PC2-C and with the positions of the predicted domains, two domain model and the of the linker region (Fig. 1D). these results we PC2-EF (Asn720–Pro797) and PC2-CC (Gly828–His927) for biophysical analysis. PC2-C a and Ca2+-induced de novo model of PC2-C predicts an all To we the structure of the PC2-C model with by were for PC2-C, PC2-EF, and PC2-CC and show to contain in with modeling results (Fig. structure using L. Nucleic Acids Res. 2004; 32: PubMed Scopus Google Scholar), was with from structural model of PC2-C for the model and for the model of PC2-C a Ca2+ binding we the Ca2+ of the of PC2-C. PC2-C and PC2-EF were in the and of PC2-EF shows an from to after Ca2+ and PC2-C from to with for PC2-C and PC2-EF with structural of PC2-C and PC2-EF structure from for PC2-CC is in with structural model of the coiled coil domain the predicted by model of PC2-C and that Ca2+ binding a conformational the EF-hand PC2-C a EF-hand by by of the EF-hand of an EF-hand motif the C-terminal cytoplasmic tail of PC2 has been Y. K. M. Y. E. J. E. Somlo S. P. 1998; 53: Full Text Full Text PDF PubMed Scopus Google Scholar), Our structural model and suggest that this EF-hand domain binds To and this Ca2+ we isothermal titration calorimetry (Fig. with a binding model that PC2-C binds Ca2+ with a of and a of whereas PC2-EF binds Ca2+ with affinity and To that residues predicted by ROBETTA model of the EF-hand domain of PC2 are directly involved in Ca2+ we created by site-directed and are predicted to be directly involved in Ca2+ to model and the a of affinity for Ca2+ (Fig. to PC2-C in a by of with in a binding of that a of PC2-C in the This result with PC2-EF, where to have all The of correlates with the affinity of the EF-hand The for Ca2+ binding the from The affinity for Ca2+ of PC2-EF PC2-C that regions of PC2-C outside of PC2-EF are involved in or of the and the of a of the C-terminal of by an as by structural model and that PC2-CC contains a coiled coil domain with a We this region of PC2-C is important for SEC was used to the and oligomerization of PC2-C, PC2-EF, and PC2-CC (Fig. PC2-C at suggesting both oligomerization and an elongated molecular Ca2+ from PC2-C the molecular by a in oligomerization or molecular by PC2-EF at with the molecular of a whereas PC2-CC at both oligomerization and an elongated To the that residues to positions a and in the PC2-CC coiled coil interface are directly PC2 oligomerization, we created PC2-CC variants with of a or residues to or V846E, M849K, I853P, I856K, V846E/I856K, or All mutations in of PC2-CC suggesting the of the of the coiled coil interface for and It is that the interface created by these a and residues was by the of charged residues or helix residues in and mutations in truncation of the coiled coil have been identified in PKD proposed coiled coil no mutations in have been structural model of PC2-C and show that PC2-C is an with PC2-CC for this the C-terminal of an with by the the oligomerization and molecular of PC2-C, PC2-EF, and PC2-CC, we performed ultracentrifugation In a be determined and multiple The a of the B. H. J. 1997; Full Text PDF PubMed Scopus Google Scholar), whereas in the to both and molecular the with a the of on the of and the in the data B. E. Sci. Scopus Google Scholar). We used a the of to (Fig. that all were of and and with and several with results suggest that PC2-CC is as an or with an elongated and that PC2-EF is with globular (Fig. A and PC2-C to be in a in which at the to be shifted to the (Fig. results are in with SEC and a model of PC2-C as an elongated oligomerization is mediated by the C-terminal coiled coil PC2-CC, identified in this PKD, PC2 is frequently truncated in PC2-C. The currently accepted domain model of PC2-C fails to explain the mechanisms by which truncations in PC2-C lead to PKD. Moreover, direct PC2 binding to involved in PKD requires residues outside this region of PC2-C. To address these discrepancies and a to investigate the role of PC2-C in PC2 function and PKD progression, we performed de novo molecular modeling and validated this model through biochemical and biophysical analysis. modeling using the ROBETTA server predicts an C-terminal to the currently accepted coiled coil domain of The of structural model with limited and results that is a previously unidentified coiled coil domain C-terminal to the EF-hand and that this is the actual coiled coil domain assumed in the literature. and SEC, we the EF-hand motif domain to be whereas the coiled coil domain of a and residues predicted to the PC2 coiled coil interface result in of PC2-CC, suggesting their in and This is in with for TRP where a C-terminal coiled coil region channel oligomerization D. Jr., 2006; Full Text Full Text PDF PubMed Scopus (124) Google Scholar), to be on PC2 Ca2+ binding to the EF-hand motif domain was using ITC, and site-directed This is the data the of a EF-hand motif Ca2+ with from to J. PubMed Scopus Google Scholar), and experiments for the EF-hand domain of PC2 suggest with micromolar the of Ca2+ predicted at the of the PC2 channel and of of or inositol 1,4,5-trisphosphate receptor at the a micromolar affinity for Ca2+ may PC2-EF to changes in Ca2+ This with the conformational changes Ca2+ a functional role for Ca2+ in PC2 We the molecular model of the C-terminal cytoplasmic tail of This model a to experiments PC2 function and the molecular basis of PKD pathogenesis and Our results the of a functional and a previously coiled coil domain in suggest that the coiled coil domain may as a PC2 oligomerization the of a coiled coil may a structural for that show the of this region for PC2 binding to PC1 and proteins. all PKD-associated PC2 truncations disrupt this PC2 coiled coil Our results a molecular for PKD We E. of the Yale for the experiments and S. Somlo, L. and S. for with