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

Abstract

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 ␤-roll and three ␣-helices. Recent evidence has demonstrated that the third (or C) ␣-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 ␤-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 Å 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 ␤-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/␤-roll interactions may also be a general feature of the mechanism of activation of other cyclic nucleotide-binding proteins.Cyclic nucleotides regulate the activity of a diverse family of proteins involved in cellular signaling. These include a transcription factor (the bacterial catabolite activating protein, CAP), the cAMP-(PKA) 1 and cGMP-dependent protein kinases (PKG) and the cyclic nucleotide-gated (CNG) ion channels involved in visual and olfactory signal transduction (1, 2). Despite obvious divergence among the effector domains of these proteins, the cyclic nucleotide binding (CNB) sites appear to share a common architecture. Solution of the crystal structures of CAP (3) and a recombinant bovine PKA RI␣ subunit (4) has demonstrated that their CNB sites are formed from an ␣-helix (A helix), an 8-stranded ␤-roll, and two more ␣-helices (B and C), with the C-helix forming the back of the binding pocket. Six residues are invariant among all members of the CAP and kinase families: three glycines involved in turns between strands of the ␤-roll, an arginine and a glutamate, each of which contact the cyclic nucleotide, and an alanine whose function is uncertain (1) (see also Fig. 1). Strikingly, these six residues are conserved in the CNG channels. Thus, it has been suggested that the invariant residues play important and conserved roles in the fol...

Double electron–electron resonance reveals cAMP-induced conformational change in HCN channels

“…The ligand-induced movement of the C-helix is widely thought to initiate the conformational changes that lead to opening of the channel pore, but the structural evidence in support of this hypothesis is equivocal (10,(24)(25)(26)(27)(28)(29). The crystal structure of the HCN2 carboxyl-terminal region in the absence of ligand shows little difference from the cyclic nucleotide-bound structure (12).…”

Distinct Structural Determinants of Efficacy and Sensitivity in the Ligand-binding Domain of Cyclic Nucleotide-gated Channels

“…Similarly, the putative ligand contact position is defined as bCNGA1 Asp 604 and homologous positions in other BDs. New X chimeras were constructed by substitution of the BD in the chimera ROON-S2 previously studied (21,22); new RO chimeras were constructed by BD substitution in the chimera RO133 previously studied (13,21,(23)(24)(25). X-bA1 and RO-bA1 in this work are synonymous with ROON-S2 and RO133, respectively.…”

Absence of Direct Cyclic Nucleotide Modulation of mEAG1 and hERG1 Channels Revealed with Fluorescence and Electrophysiological Methods

“…Mutations of the Arg residue to Lys or Trp in PKA (54,55), to several different residues, including Ala, in CAP (52,53,56), and to Gln in MlotiK1 channels (57) abolished cAMP binding. Mutation of the Arg residue to Ala in MlotiK1 and HCN2 channels, and to neutral and negatively charged residues in CNGA1 channels drastically decreased apparent cyclic nucleotide binding affinity (51,58,59).…”