Structural and biophysical characterization of polycystin family cation channel cytoplasmic regulatory domains
Abstract
The Polycystin sub-family of transient receptor potential (TRP) channels is composed of two membrane-bound classes: Polycystic Kidney Disease 1-like (PKD1-like) proteins PKD1, PKD1L2, and PKD1L3, which act as sensors of changes to the extracellular milieu, and Polycystic Kidney Disease 2-like (PKD2-like) proteins PKD2 and PKD2L1 that act as Ca2+ permeable cation channels. Homomeric PKD2 channels reside in the endoplasmic reticulum, where they assist in regulation of intracellular Ca2+ stores, while PKD2L1 channels in neurons lining the spinal column are implicated in monitoring and responding to small changes in the pH of cerebrospinal fluid. The PKD2L1/PKD1L3 complex perceives sour taste, while disruption of the PKD2/PKD1 complex, responsible for mechanosensation in kidney epithelial cell primary cilia, leads to development of autosomal dominant polycystic kidney disease (ADPKD). The carboxyl-terminal regulatory domains (CRDs) of PKD2 and PKD2L1 play a multitude of roles that affect subcellular trafficking, protein-protein interactions, signaling, and channel conductance, and mutations or truncations of the CRDs can have a significant impact on complex assembly, function, and subcellular localization. Due to their importance, the studies detailed herein were conducted to characterize the biochemical and biophysical properties of PKD2 and PKD2L1 CRDs, address the stoichiometry of assembly, and determine the identity and structure of the region responsible for oligomerization. This study has found the isolated EF-hands of PKD2 and PKD2L1 to bind Ca 2+ with low micromolar affinity (Kd values of 2.8 and 0.51 μM, respectively). Additionally, it was found that the CRDs of PKD2 and PKD2L1 assemble as trimers possessing an elongated shape, with a newly defined coiled-coil region mediating assembly. The 2.7 Å resolution crystal structure of the PKD2L1 trimerization domain revealed that oligomerization is driven by beta-branched residues in the “a” and “d” positions of the heptad repeat. When these residues are mutated to alanine, the oligomerization state of the isolated PKD2L1 CRD (which remains exclusively trimeric in solution at concentrations approaching 750 nM) shifts from trimeric to monomeric. This monomeric variant can be expressed and purified, and binds Ca2+ with an affinity similar to that of wild-type PKD2L1 CRD, but removal of ligand leads to a significant (38 %) reduction of alpha-helical content. Such a significant drop in secondary structure suggests that the role of the observed high-affinity CRD trimerization may be to lend stability in absence of ligand.
Degree
Ph.D.
Advisors
Yernool, Purdue University.
Subject Area
Molecular biology|Cellular biology|Biophysics
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