Nucleotides bind to the C-terminus of ClC-5
| Autor: | Hsin-Hen Kuo, Charles M. Deber, Fiona L. L. Stratford, Leigh Wellhauser, Canhui Li, Winnie Luong, Ling-Jun Huan, Christine E. Bear, Mohabir Ramjeesingh |
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| Rok vydání: | 2006 |
| Předmět: |
Protein Denaturation
CBS domain Gene Expression Biochemistry Protein Structure Secondary Protein structure Adenosine Triphosphate Chloride Channels Protein purification Humans Nucleotide Molecular Biology Protein secondary structure chemistry.chemical_classification Adenosine Triphosphatases Chemistry urogenital system C-terminus Circular Dichroism Hydrolysis Temperature Cell Biology Adenosine Monophosphate Recombinant Proteins Cytoplasm Chloride channel Research Article |
| Zdroj: | The Biochemical journal. 398(2) |
| ISSN: | 1470-8728 |
| Popis: | 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 β-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 α-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 μM 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. |
| Databáze: | OpenAIRE |
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