The Structure of the Prokaryotic Cyclic Nucleotide-Modulated Potassium Channel MloK1 at 16 Å Resolution

Abstract

The gating ring of cyclic nucleotide-modulated channels is proposed to be either a two-fold symmetric dimer of dimers or a four-fold symmetric tetramer based on high-resolution structure data of soluble cyclic nucleotide-binding domains and functional data on intact channels. We addressed this controversy by obtaining structural data on an intact, full-length, cyclic nucleotide-modulated potassium channel, MloK1, from Mesorhizobium loti, which also features a putative voltage-sensor. We present here the 3D single-particle structure by transmission electron microscopy and the projection map of membrane-reconstituted 2D crystals of MloK1 in the presence of cAMP. Our data show a four-fold symmetric arrangement of the CNBDs, separated by discrete gaps. A homology model for full-length MloK1 suggests a vertical orientation for the CNBDs. The 2D crystal packing in the membrane-embedded state is compatible with the S1-S4 domains in the vertical "up" state.

Structure of TRPV1 channel revealed by electron cryomicroscopy

“…Importantly, the structural and functional integrity of the recombinant protein was established by gel-filtration chromatography and ligand-gated Ca 2ϩ flux assays. We find that the TRPV1 structure is consistent with those of other tetrameric six-transmembrane-segment channels, conforming to a two-domain motif referred to as a ''hanging gondola'' (17)(18)(19)(20)(21).…”

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

“…A comparison with CNBD crystal structures from both proteins in the ligand-bound state (Clayton et al, 2004;Rehmann et al, 2008) shows that the conformational change of the CNBD upon ligand-binding contradicts this novel interface, suggesting that the dissociation of CNBD dimers upon ligand-binding could be an alternative mechanism of ligand gating in the MloK1 channel. This observation is consistent with the previously reported low resolution electron microscopy structure of the MloK1 channel in the presence of cAMP, where the CNBDs are completely separated by discrete spacing (Chiu et al, 2007).…”

“…The tetrameric organization of the channel was confirmed by a cryo-electron microscopy structure of the MloK1 channel (Chiu et al, 2007) and a crystal structure of the MloK1 transmembrane segments in the closed state with a resolution of 3.1 Å . However, the CNBDs were not resolved in the crystal structure.…”

“…However, a cryo-electron microscopy structure of the full-length MloK1 channel in the presence of cAMP at 16 Å resolution revealed a fourfold symmetric arrangement of the CNBDs separated by discrete gaps (Chiu et al, 2007). Ligand-binding studies showed that the whole channel and the monomeric CNBD bind cAMP non-cooperatively with similar affinity (Cukkemane et al, 2007).…”

“…Although ligand-induced conformational changes could not be observed for the wild-type MloK1 channel, the CNBD R348A mutant, which has a lower affinity for cAMP and thereby allows the preparation of the ligand-bound and -free state (Clayton et al, 2004;Cukkemane et al, 2007;Altieri et al, 2008;Peuker et al, 2013), was used to study conformational changes of the CNBDs upon ligand-binding and -dissociation. In the ligand-bound state, the CNBDs are arranged with a four-fold symmetry displayed by a cryo-electron microscopy structure (Chiu et al, 2007). The AFM topographs (Mari et al, 2011) showed that upon ligand-binding and dissociation a reversible conformational change of the mutant CNBDs occurs.…”