A state-independent interaction between ligand and a conserved arginine residue in cyclic nucleotide-gated channels reveals a functional polarity of the cyclic nucleotide binding site.

Activation of cyclic nucleotide-gated channels is thought to involve two distinct steps: a recognition event in which a ligand binds to the channel and a conformational change that both opens the channel and increases the affinity of the channel for an agonist. Sequence similarity with the cyclic nucleotide-binding sites of cAMP- and cGMP-dependent protein kinases and the bacterial catabolite activating protein (CAP) suggests that the channel ligand binding site consists of a beta-roll and three alpha-helices. Recent evidence has demonstrated that the third (or C) alpha-helix moves relative to the agonist upon channel activation, forming additional favorable contacts with the purine ring. Here we ask if channel activation also involves structural changes in the beta-roll by investigating the contribution of a conserved arginine residue that, in CAP and the kinases, forms an important ionic interaction with the cyclized phosphate of the bound ligand. Mutations that conserve, neutralize, or reverse the charge on this arginine decreased the apparent affinity for ligand over four orders of magnitude but had little effect on the ability of bound ligand to open the channel. These data indicate that the cyclized phosphate of the nucleotide approaches to within 2-4 A of the arginine, forming a favorable ionic bond that is largely unaltered upon activation. Thus, the binding site appears to be polarized into two distinct structural and functional domains: the beta-roll stabilizes the ligand in a state-independent manner, whereas the C-helix selectively stabilizes the ligand in the open state of the channel. It is likely that these distinct contributions of the nucleotide/C-helix and nucleotide/beta-roll interactions may also be a general feature of the mechanism of activation of other cyclic nucleotide-binding proteins.