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

Borries Demeler,Titus J. Boggon,Barbara E. Ehrlich,Andjelka Ćelić,Edward T. Petri

doi:10.1074/jbc.m802743200

Abstract

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.

Highlights

Polycystic kidney disease (PKD)4 is among the most common life-threatening inherited disorders, with clinical consequences characterized by renal and hepatic cysts [1]
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
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)

Summary

EXPERIMENTAL PROCEDURES

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 Purified polycystin-2 fragments were eluted in Buffer A ϩ 500 mM imidazole and applied to a Superdex 200 SEC column equilibrated in Buffer A. Coordinates for the ROBETTA models are included as supplemental data. Proteins were stripped of Ca2ϩ with 100 mM EDTA and desalted in 25 mM Tris, 250 mM NaCl, pH 7.5. Size Exclusion Chromatography—PC2-C, PC2-EF, or PC2-CC (0.5 ml each) was applied to a Superdex 200 analytical grade SEC column (GE Healthcare) equilibrated in 25 mM Tris, 250 mM NaCl, pH 8.0, at 0.1 ml/min. Equilibrium data were fit to multiple models, and the best chosen based on fitting statistics and visual inspection of the residual run patterns. Samples were analyzed in a buffer containing 300 mM NaCl. Sedimentation velocity experiments were performed at 20 °C and 60,000 rpm. Lamm equation [35] combined with the enhanced van HoldeWeischet method as implemented in UltraScan [36]

RESULTS AND DISCUSSION

Proteolysis Validates the Presence and Relative Positions of Two

Mediated by an Extended Coiled Coil Domain as Shown by Size