The ubiquitous CBS domains, which are found as part of cytoplasmic domains in the ClC family of chloride channels and transporters, have previously been identified as building blocks for regulatory nucleotide-binding sites. Here we report the structures of the cytoplasmic domain of the human transporter ClC-5 in complex with ATP and ADP. The nucleotides bind to a specific site in the protein. As determined by equilibrium dialysis, the affinities for ATP, ADP and AMP are in the high micromolar range. Point mutations that interfere with nucleotide binding change the transport behavior of a ClC-5 mutant expressed in Xenopus laevis oocytes. Our results establish the structural and energetic basis for the interaction of ClC-5 with nucleotides and provide a framework for future investigations.
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X-ray data were collected (MDC Berlin) at the Swiss Light Source of the Paul Scherrer Institute. We thank T. Jentsch (MDC Berlin) for providing the ClC-5 clone, S. Chesnov and P. Hunziker for help with mass spectrometry, B. Blattmann for help with crystal screening, P. Lindner for advice on equilibrium binding assays, E. Hänsenberger for preparation of the Xenopus oocytes, the staff of the X06SA beamline for their support during data collection and R. MacKinnon for comments on the manuscript. This work was supported by a grant from the Swiss National Science Foundation and the National Center of Competence in Research in Structural Biology program. S.M. is affiliated with the Molecular Life Sciences PhD Program of the University/ETH Zürich.
The authors declare no competing financial interests.
Sedimentation velocity data for the ClC-5 cytoplasmic domain. (PDF 336 kb)
Stereo view of experimental density of the nucleotide-binding region in the ClC-5 domain–ATP complex. (PDF 334 kb)
Competition of bound radiolabeled ATP. (PDF 11 kb)
Two-electrode voltage-clamp recordings of human ClC-5 in Xenopus oocytes. (PDF 179 kb)
Two-electrode voltage-clamp recordings of human ClC-5 mutants in Xenopus oocytes. (PDF 175 kb)
Two-electrode voltage-clamp recordings of human ClC-5 double mutants in Xenopus oocytes. (PDF 176 kb)
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