title: ATP induces conformational changes in the carboxyl-terminal region of ClC-5.
abstract: ATP binding enhances the activity of ClC-5, the transporter mutated in Dent disease, a disease affecting the renal proximal tubule. Previously, the ATP binding site was revealed in x-ray crystal structures of the cytoplasmic region of this membrane protein. Disruption of this site by mutagenesis (Y617A-ClC-5) reduced the functional expression and ATP-dependent regulation of the full-length transporter in Xenopus oocytes. However, insight into the conformational changes underlying ATP-dependent regulation is lacking. Here, we show that ATP binding induces a change in protein conformation. Specifically, small angle x-ray scattering experiments indicate that ATP binding promotes a clamp-like closure of the isolated ClC-5 carboxyl-terminal region. Limited proteolysis studies show that ATP binding induces conformational compaction of the carboxyl-terminal region in the intact membrane protein as well. In the context of fibroblasts and proximal tubule epithelial cells, disruption of the ATP binding site in full-length ClC-5 (Y617A-ClC-5) led to a defect in processing and trafficking out of the endoplasmic reticulum. These latter findings account for the decrease in functional expression previously reported for this ATP-binding mutant and prompt future study of a model whereby conformational compaction caused by ATP binding promotes biosynthetic maturation.
authors.affiliation: Programme of Molecular Structure and Function, Research Institute, the Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
authors.forename: Leigh
authors.initials: L
authors.lastname: Wellhauser
authors.forename: Cesar
authors.initials: C
authors.lastname: Luna-Chavez
authors.forename: Christina
authors.initials: C
authors.lastname: D'Antonio
authors.forename: John
authors.initials: J
authors.lastname: Tainer
authors.forename: Christine E
authors.initials: CE
authors.lastname: Bear
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D057973:Dent Disease / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016296:Mutagenesis; D020125:Mutation, Missense; D009865:Oocytes; D010449:Peptide Mapping; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000235:genetics; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: C498610:CIC protein, human; D012097:Repressor Proteins; D000255:Adenosine Triphosphate