A functioning chimera of the cyclic nucleotide-binding domain from the bovine retinal rod ion channel and the DNA-binding domain from catabolite gene-activating protein

S. P. Scott, I. T. Weber, R. W. Harrison, J. Carey, J. C. Tanaka

Research output: Contribution to journalArticle

17 Scopus citations

Abstract

The eukaryotic cyclic nucleotide-gated (CNG) ion channels are a family of large membrane proteins activated by cytoplasmic cGMP or cAMP. Their cyclic nucleotide-binding domain is structurally homologous with that of the catabolite gene-activator protein (CAP), a soluble Escherichia coli transcription factor. Differences in ligand activation among sensory channels suggest differences in the underlying molecular mechanisms of signal readout. To study the structural, functional, and conformational consequences of nucleotide binding, we fused the cyclic nucleotide-binding domain from the bovine retinal rod CNG channel α subunit (Brα) to the DNA-binding domain from CAP. The chimera forms a soluble dimer that binds both cGMP and cAMP with association constants of 3.7 × 104 M-1for [3H]cGMP and 3.1 × 104 M-1 for [3H]cAMP. The binding of cAMP, but not cGMP, exposes a chymotrypsin cleavage site in the chimera at a position similar to the site in the CAP exposed by cAMP binding. At high cAMP concentrations, a biphasic pattern of cleavage is seen, suggesting that the low-affinity cAMP binding sites are also occupied. Cyclic AMP promotes specific binding to a DNA fragment encoding the lac operator region; the Kd for the protein-DNA binding is ∼200 nM, which is 2-fold higher than the Kd for CAP under identical conditions. A 7 Å crystal structure shows that the overall secondary and tertiary structure of Brα/CAP is the same as that of CAP with two cAMP molecules bound per dimer. The biochemical characterization of the chimera suggests it will be a useful system for testing hypotheses about channel activation, providing further insight into channel function.

Original languageEnglish (US)
Pages (from-to)7464-7473
Number of pages10
JournalBiochemistry
Volume40
Issue number25
DOIs
StatePublished - Jun 26 2001

All Science Journal Classification (ASJC) codes

  • Biochemistry

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