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.
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 alpha subunit (Bralpha) 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 x 10(4) M(-1) for [(3)H]cGMP and 3.1 x 10(4) M(-1) for [(3)H]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 K(d) for the protein-DNA binding is approximately 200 nM, which is 2-fold higher than the K(d) for CAP under identical conditions. A 7 A crystal structure shows that the overall secondary and tertiary structure of Bralpha/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.
Scott
S P
SP
Departments of Biology, Chemistry, and Computer Science, Georgia State University, Atlanta, Georgia 30303, USA.
Weber
I T
IT
Harrison
R W
RW
Carey
J
J
Tanaka
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D018360:Crystallography, X-Ray; D000242:Cyclic AMP / Q000378:metabolism; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D054815:Cyclic Nucleotide-Gated Cation Channels; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000138:chemical synthesis; D006868:Hydrolysis; D007473:Ion Channels / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D012374:Rod Cell Outer Segment / Q000502:physiology*; D012995:Solubility; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; D054815:Cyclic Nucleotide-Gated Cation Channels; D004268:DNA-Binding Proteins; D007473:Ion Channels; D011993:Recombinant Fusion Proteins; D004247:DNA; D000242:Cyclic AMP; D002918:Chymotrypsin