The structure of the prokaryotic cyclic nucleotide-modulated potassium channel MloK1 at 16 A 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.

Figure 1

(a) Proposed topology of one MloK1 subunit. Each subunit consists of an N-terminal transmembrane domain and a C-terminal cyclic nucleotide-binding domain (CNBD). Helices S1-S4 (blue) are the putative voltage sensors, and helices S5, S6, and the pore helix (light orange) form the pore region. CNBD is shown in ribbon representation, bound cAMP is shown space-filled in cpk. (b) Sequence alignment of the transmembrane domains of MloK1 (Mesorhizobium loti), Kv1.2 (Rattus norvegicus) and Shaker (Drosophila melanogaster). The secondary structures of Kv1.2 are indicated. Residues of similar chemical characteristics are yellow, identical residues are light blue, and positively charged amino acids (arginine, lysine) in S4 are red.

Figure 2

Single-particle TEM imaging of MloK1. (a) Electron micrograph of negatively-stained MloK1 particles. Detergent-solubilized MloK1 are bright on dark background. Scale bar 50 nm. (b) Selected class views and particle images of the MloK1 single particles. The first row is individual particle images from three classes in columns, the second row shows the corresponding class averages, and the last row is the p4-symmetrized 3D reconstruction density projected in the same direction. The width of the square panels is 16 nm. (c) Fourier-shell correlation (FSC) plot of the final 3D reconstruction, indicating a resolution of 16.3 Å (arrow, 0.5 criterion (Böttcher et al., 1997)). (d) The 3D reconstruction from 5018 single-particle images. Left, the proposed top-view from the extracellular side. Center, the bottom view, showing the four CNBDs. Right, the side view. The approximate membrane plane is indicated by the dotted lines. Scale bar 2 nm. Inside each density we show the docking of the high-resolution structures of Kv1.2 (PDB code: 2A79) and the MloK1 CNBD (PDB code: 1VP6) into the MloK1 3D reconstruction. The α-helices are in blue, β-strands in yellow, and loops in green. Red balls represent K ions in the pore.

Figure 3

Arrangement of the CNBDs in the constructed model. (a) A vertical arrangement of the CNBDs fits best into the determined 3D structure. (b) The horizontal arrangement of the CNBDs as in the HCN2 CNBD crystal structure cannot satisfyingly be docked into the 3D volume. S1-S4 helices, blue; S5-S6, yellow, CNBD, red. Scale bar 5 nm.

Figure 4

Two-dimensional membrane crystal of MloK1. (a) Electron micrograph of a negatively stained 2D crystal of membrane-reconstituted MloK1. The bright lines are the edges of the membrane crystals; the square lattice of the crystal is clearly visible. Scale bar 200 nm. (b) Typical calculated power spectrum, showing diffraction spots at 2 nm resolution before image processing. (c) The non-symmetrized reconstruction, showing 2×2 unit cells in p42₁2 symmetry. Unit cell dimensions are a=b=129Å, γ=90°. Protein is white in (a) and (c).

Figure 5

Projection maps of evenly (a) and unevenly (b) stained 2d crystals of MloK1 after image processing. A difference map (c) between the two shows the contours of one surface only. Insets in (a) to (c) are cartoons that depict the staining level. (d) Arrangement of the single particle reconstruction in the 2d crystal-like alternative orientations with overlaid X-ray structures of Kv1.2 (S1-S4, red; S5-S6, yellow) and MloK1 CNBD (blue).

Figure 6

The homology model with “vertically” oriented paddles fit to the 2D crystal projection map. (a) 2×2 unit cell arrangement. Protein is in white. (b) Side view of the two tetramers that are in (a) indicated by the dotted line and seen as indicated by the arrow. Scale bar 10 nm. S1-S4 helices, blue; S5-S6, yellow, CNBD, red.