Abstract
PC2 (polycystin-2) forms a Ca2+ -permeable channel in the cell membrane and its function is regulated by cytosolic Ca2+ levels. Mutations in the C-terminal tail of human PC2 (HPC2 Cterm) lead to autosomal dominant polycystic kidney disease. The HPC2 Cterm protein contains a Ca2+ -binding site responsible for channel gating and function. To provide the foundation for understanding how Ca2+ regulates the channel through the HPC2 Cterm, we characterized Ca2+ binding and its conformational and dynamic responses within the HPC2 Cterm. By examining hydrogen-deuterium (H-D) exchange profiles, we show that part of the coiled-coil domain in the HPC2 Cterm forms a stable helix bundle regardless of the presence of Ca2+. The HPC2 L1EF construct contains the Ca2+ -binding EF-hand and the Nterminal linker 1 region without the downstream coiled coil. We show that the linker stabilizes the Ca2+ -bound conformation of the EF-hand, thus enhancing its Ca2+ -binding affinity to the same level as the HPC2 Cterm. In comparison, the coiled coil is not required for the high-affinity binding. By comparing the conformational dynamics of the HPC2 Cterm and HPC2 L1EF with saturating Ca2+ , we show that the HPC2 Cterm and HPC2 L1EF share a similar increase in structural stability upon Ca2+ binding. Nevertheless, they have different profiles of H-D exchange under non-saturating Ca2+ conditions, implying their different conformational exchange between the Ca2+ -bound and -unbound states. The present study, for the first time, provides a complete map of dynamic responses to Ca2+ -binding within the full-length HPC2 Cterm. Our results suggest mechanisms for functional regulation of the PC2 channel and PC2's roles in the pathophysiology of polycystic kidney disease.
Original language | English (US) |
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Pages (from-to) | 285-296 |
Number of pages | 12 |
Journal | Biochemical Journal |
Volume | 473 |
Issue number | 3 |
DOIs | |
State | Published - Feb 1 2016 |
Keywords
- Ca -binding proteins
- Hydrogen-deuterium exchange mass spectrometry
- Isothermal titration calorimetry
- Nuclear magnetic resonance
- Polycystin-2
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology