Structural and Functional Regulation of the Human Chloride/Proton ClC-5 by ATP and Scaffold NHERF2 Interactions

• Main Author: Wellhauser, Leigh Anne
• Other Authors: Bear, Christine
• Language: en_ca
• Published: 2010
• Subjects: ClC-5; CBS Domains; ATP Binding Site; NHERF2; SAXS; Thermal Stability; Photoaffinity Labelling; Endocytosis; Internal PDZ Binding Motifs; 0487
• Online Access: http://hdl.handle.net/1807/32054

The chloride/proton antiporter ClC-5 is primarily expressed in the kidney where it aids in re-absorption of proteins from the glomerular filtrate. Functional disruption of ClC-5 causes Dent’s Disease – a renal condition characterized by proteinuria and kidney failure in a third of all cases. The majority of disease-causing mutations translate into premature truncations of the carboxy-terminal (Ct) region of ClC-5 and are predicted to disrupt the protein-protein interactions mediated by this domain. In this thesis, direct ATP binding to the two cystathionine β-synthase (CBS) domains of ClC-5 was demonstrated. ATP binding enhanced the global compactness of the ClC-5 Ct region likely through a clamping motion of the CBS domains around the nucleotide. Along with ATP, the sodium proton exchange regulatory factor 2 (NHERF2) also binds ClC-5; however, the molecular mechanism behind this interaction was unknown as ClC-5 lacked the PDZ binding motif traditionally localized at the Ct end of bait proteins. Here, we also identified a class I PDZ binding motif (657-660; TSII) within the internal sequence of ClC-5. Despite the buried position of this motif in the Ct peptide’s X-ray crystal structure (PDB: 2J9L), the high propensity of this region for dynamic flexibility prompted us to test whether it could mediate NHERF2 interactions. In support of this hypothesis, we demonstrated that the motif is transiently available to interact directly with NHERF2 in vivo and to enable an enhancement in receptor-mediated endocytosis in mammalian cells. Collectively, these results gave further evidence that the intracellular Ct region of ClC-5 serves as a hub to mediate interactions essential for its maturation, stability, and trafficking in renal epithelium, as well as providing further insights into the molecular basis of Dent’s Disease.