Structure of the EF-hand domain of polycystin-2 suggests a mechanism for Ca ²⁺ -dependent regulation of polycystin-2 channel activity

Abstract

The C-terminal cytoplasmic tail of polycystin-2 (PC2/TRPP2), a Ca 2+ -permeable channel, is frequently mutated or truncated in autosomal dominant polycystic kidney disease. We have previously shown that this tail consists of three functional regions: an EF-hand domain (PC2-EF, 720–797), a flexible linker (798–827), and an oligomeric coiled coil domain (828–895). We found that PC2-EF binds Ca 2+ at a single site and undergoes Ca 2+ -dependent conformational changes, suggesting it is an essential element of Ca 2+ -sensitive regulation of PC2 activity. Here we describe the NMR structure and dynamics of Ca 2+ -bound PC2-EF. Human PC2-EF contains a divergent non-Ca 2+ -binding helix-loop-helix (HLH) motif packed against a canonical Ca 2+ -binding EF-hand motif. This HLH motif may have evolved from a canonical EF-hand found in invertebrate PC2 homologs. Temperature-dependent steady-state NOE experiments and NMR R 1 and R 2 relaxation rates correlate with increased molecular motion in the EF-hand, possibly due to exchange between apo and Ca 2+ -bound states, consistent with a role for PC2-EF as a Ca 2+ -sensitive regulator. Structure-based sequence conservation analysis reveals a conserved hydrophobic surface in the same region, which may mediate Ca 2+ -dependent protein interactions. We propose that Ca 2+ -sensing by PC2-EF is responsible for the cooperative nature of PC2 channel activation and inhibition. Based on our results, we present a mechanism of regulation of the Ca 2+ dependence of PC2 channel activity by PC2-EF.

Calcium-induced Conformational Changes in C-terminal Tail of Polycystin-2 Are Necessary for Channel Gating

“…The resulting D max values are in agreement with the lengths of the previously determined trimeric crystal structure of the PC2 coiled coil domain (Protein Data Bank code 3HRN) (7) (ϳ90 Å) and the NMR structure of PC2-EF (Protein Data Bank code 2K6Q) (5) (ϳ40 Å), especially when one considers that these structures comprise only 53% of the 265 PC2-C residues (141 residues; 78 from the EF-hand and 63 from the coiled coil). These significant reductions in R g and D max in the presence of excess Ca 2ϩ correlate with a compression of the overall structure of PC2-C that could be mediated by Ca 2ϩ -induced folding and interdomain interactions, as we previously suggested (5).…”

“…1C). Previously, we showed by NMR that PC2-EF is an atypical EF-hand domain containing only one Ca 2ϩ -binding site (5) and that PC2-EF undergoes an increase in ␣-helicity from 27 to 45% upon the addition of Ca 2ϩ by circular dichroism (4). Therefore, we hypothesize that these macromolecular envelope changes upon the addition of Ca 2ϩ to PC2-EF result from a transition from a Ca 2ϩ -unbound to Ca 2ϩ -bound state, analogous to conformational changes observed in other EF-hand proteins on binding Ca 2ϩ (37).…”

Molecular Architecture and Subunit Organization of TRPA1 Ion Channel Revealed by Electron Microscopy

“…However an atomic resolution structure for a full-length TRP channel is not yet available. Isolated domains of TRP channels have been solved by x-ray crystallography or NMR, including the ␣-kinase domain of TRPM7 (10), the ankyrin repeat domains from the TRPV subfamily of proteins (11)(12)(13)(14)(15), the C-terminal cytoplasmic coiled-coil domains of TRPM7 and TRPP2 (16,17), and the EF hand domain of TRPP2 (18,19). These partial atomic structures are useful for understanding channel regulation/modulation and enhance the interpretation of moderate resolution fulllength TRP channel structures (20).…”

Cation-driven Optical Properties of Artificial Luciferases

“…1(A) and S2), which appears at the corresponding sites of the known EF-hand motif of Ca 2+ -binding proteins like coelenterazine-binding protein (CBP). 12 The structure is rare among non-Ca 2+ -binding proteins. Point mutations in the amino acids (E150Y, A182Y, E150W, and A182W) in the proposed EF-hand-like structure completely destroyed the optical intensities of ALuc25 (Fig.…”