title: Nucleotides bind to the C-terminus of ClC-5.
abstract: Mutations in ClC-5 (chloride channel 5), a member of the ClC family of chloride ion channels and antiporters, have been linked to Dent's disease, a renal disease associated with proteinuria. Several of the disease-causing mutations are premature stop mutations which lead to truncation of the C-terminus, pointing to the functional significance of this region. The C-terminus of ClC-5, like that of other eukaryotic ClC proteins, is cytoplasmic and contains a pair of CBS (cystathionine beta-synthase) domains connected by an intervening sequence. The presence of CBS domains implies a regulatory role for nucleotide interaction based on studies of other unrelated proteins bearing these domains [Ignoul and Eggermont (2005) Am. J. Physiol. Cell Physiol. 289, C1369-C1378; Scott, Hawley, Green, Anis, Stewart, Scullion, Norman and Hardie (2004) J. Clin. Invest. 113, 274-284]. However, to date, there has been no direct biochemical or biophysical evidence to support nucleotide interaction with ClC-5. In the present study, we have expressed and purified milligram quantities of the isolated C-terminus of ClC-5 (CIC-5 Ct). CD studies show that the protein is compact, with predominantly alpha-helical structure. We determined, using radiolabelled ATP, that this nucleotide binds the folded protein with low affinity, in the millimolar range, and that this interaction can be competed with 1 muM AMP. CD studies show that binding of these nucleotides causes no significant change in secondary structure, consistent with a model wherein these nucleotides bind to a preformed site. However, both nucleotides induce an increase in thermal stability of ClC-5 Ct, supporting the suggestion that both nucleotides interact with and modify the biophysical properties of this protein.
authors: Programme in Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8.
authors: Leigh
authors: L
authors: Wellhauser
authors: Hsin-Hen
authors: HH
authors: Kuo
authors: Fiona L L
authors: FL
authors: Stratford
authors: Mohabir
authors: M
authors: Ramjeesingh
authors: Ling-Jun
authors: LJ
authors: Huan
authors: Winnie
authors: W
authors: Luong
authors: Canhui
authors: C
authors: Li
authors: Charles M
authors: CM
authors: Deber
authors: Christine E
authors: CE
authors: Bear