Structural Basis of Ligand Activation in a Cyclic Nucleotide Regulated Potassium Channel

Abstract

Here we describe the initial functional characterization of a cyclic nucleotide regulated ion channel from the bacterium Mesorhizobium loti and present two structures of its cyclic nucleotide binding domain, with and without cAMP. The domains are organized as dimers with the interface formed by the linker regions that connect the nucleotide binding pocket to the pore domain. Together, structural and functional data suggest the domains form two dimers on the cytoplasmic face of the channel. We propose a model for gating in which ligand binding alters the structural relationship within a dimer, directly affecting the position of the adjacent transmembrane helices.

Structural snapshot of cyclic nucleotide binding domains from cyclic nucleotide-sensitive ion channels

“…The length of the C-linker region in MloK1 that connects the CNBD to S6 is noticeably shorter (∼20 residues) when compared with the mammalian CNG and HCN channels (∼80 residues). Crystal structures of the mutant and wild-type CNBD in the ligand-free and -bound state, respectively, revealed insights into the conformational rearrangements in the CNBD upon ligand-binding (Clayton et al, 2004;Altieri et al, 2008). The structure of the MloK1 CNBD shows the characteristic properties similar to the HCN2 CNBD structure (Figures 1 and 2), consisting of a β roll core topped by a bundle of four α-helices and a short PBC helix (Zagotta et al, 2003).…”

“…One key residue, R591 of HCN2 located between the PBC helix and β7, interacts directly with the negatively charged exocylic oxygen of the phosphate group. This arginine residue together with its interaction is conserved (Figure 4) and mutation of this residue in CNG, HCN and MloK1 channels decrease the affinity for cyclic nucleotides (Tibbs et al, 1998;Chen et al, 2001;Clayton et al, 2004; Altieri et al, 2008;Harzheim et al, 2008;Schünke et al, 2009). The purine ring interacts through stacking and hydrogen-bond interactions with additional residues located in both the β roll and helix αC.…”

“…The prokaryotic K + -selective CNG channel MloK1 has been identified in the bacterium Mesorhizobium loti (Clayton et al, 2004;Nimigean et al, 2004) and shares a similar topology with eukaryotic CNG and HCN channels (Figure 1). The channel consists of four identical subunits, each exhibiting six transmembrane segments (S1-S6), a 'GYG' signature sequence for K + selectivity and a conserved CNBD.…”

“…A detailed structurefunction analysis of CNG and HCN channel CNBDs was initiated (see information given in Table S1) when the first CNBD crystal structures of the eukaryotic HCN2 and prokaryotic MloK1 channel were reported (Zagotta et al, 2003;Clayton et al, 2004).…”

“…Addressing the question whether the CNBD of eukaryotic HCN or CNG channels undergo structural rearrangements upon ligand-binding in a manner that differs to the bacterial MloK1 CNBD (Clayton et al, 2004;Altieri et al, 2008;Schünke et al, 2009;Mari et al, 2011;Schünke et al, 2011) is not possible, because only structural information of one ligand-free eukaryotic CNBD is available (Taraska et al, 2009) in contrast to multiple structures in the ligandbound state (see Table S1). …”