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. 2018 Jul 20;7:e39775. doi: 10.7554/eLife.39775

Figure 6. Ligand binding in the SthK CNBDs.

(A) cAMP bound in anti configuration to the binding pocket. (B) cGMP bound in the syn configuration having similar interaction with SthK. (C) Density maps (grey mesh) for cAMP (purple, anti) and cGMP (cyan, syn) showing the ligand fit. The corresponding difference maps (blue mesh) between ligand-bound and apo experimental density maps confirmed ligand binding. Densities shown are at a contour level of 5.5 σ. (D) Overlay between the cAMP-bound SthK CNBD from cryo-EM (green) and from crystallography (grey, PDB:4D7T). (E) Overlay between cGMP-bound SthK CNBD from cryo-EM (green) and from crystallography (wheat, PDB:4D7S). Insert shows potential clash of the C-helix (L422, highlighted in red) from the crystal structure with the cGMP in the syn configuration. (F) Overlay between apo SthK (green) and apo HCN2 (purple) CNBDs showing identical conformations.

Figure 6.

Figure 6—figure supplement 1. Comparisons between CNBDs of SthK and related channels.

Figure 6—figure supplement 1.

(A) Side-by-side comparison of cGMP-bound SthK CNBD from cryo-EM (green) and X-ray crystallography for the isolated domain (wheat). The green is in the resting and the wheat is in the activated conformation as shown by the C-helix position. cGMP configuration is in syn (green) and anti (wheat). (B) Overlays between apo SthK (green), apo HCN1 (yellow), and cAMP HCN1 (blue) CNBDs (left) and siphon regions (right). The C-helices are visible in lower, intermediate and upper position, respectively and the siphon loops are visible correspondingly in a lower, intermediate, and upper position. (C) Overlay between CNBDs of cyclic nucleotide-modulated ion channels shows two major conformational states of the C-helix (depicted as a cylinder), either in lower (resting) conformation or upper (activated) conformation. C-helix of apo HCN1 (yellow) is in an intermediate conformation, but closer to the upper (activated) conformation. (E) Overlays between siphon regions of apo SthK (green, left) and apo HCN2 (purple), showing close to identical conformations. The overlays between cryo-EM SthK (green; cAMP (middle) and cGMP (right)) and the crystal structure SthK (grey and wheat, cAMP and cGMP, respectively) siphons show big differences, corresponding to the large differences between resting and activated CNBDs. PDB codes: apo SthKEM (6CJQ), cAMP SthKEM (6CJU), cGMP SthKEM (6CJT), cAMP SthKXRAY (4D7T), cGMP-SthKXRAY (4D7S), apo HCN1 (5U6O), cAMP HCN1 (5U6P), TAX-4 (5H3O), LliK (5V4S), apo HCN2 (5JON), cAMP HCN2 (1Q43), cGMP HCN2 (1Q3E), MolK1 (6EO1).
Figure 6—figure supplement 2. Difference density maps for cyclic nucleotides in the CNBDs of SthK.

Figure 6—figure supplement 2.

(A) ‘Omit’ difference map (magenta mesh) between the experimental density maps for cAMP-bound SthK and a theoretical map built from the atomic model from which the ligand was deleted (pdb 6CJU). (B) ‘Omit’ difference map (cyan mesh) between the experimental density maps for cGMP-bound SthK and a theoretical map built from the atomic model from which the ligand was deleted (pdb 6CJT). In both cases, only the region of the binding pocket is shown for clarity. cAMP and cGMP are shown in magenta and cyan sticks, respectively, and part of the binding site in green cartoon (C) Difference maps (magenta mesh) between the experimental density map for cAMP-bound SthK and a theoretical map built from the atomic model where cAMP is in anti configuration (top), or syn configuration (bottom). Positive density in each case in a different location suggests that the experimental cAMP density is a mix of both syn and anti configurations. (D) Difference maps (cyan mesh) between the experimental density map for cGMP-bound SthK and a theoretical map built from the atomic model where cGMP is in syn configuration (top), or anti configuration (bottom). Positive density in only the anti case suggests that the experimental cGMP density is composed of only the syn configuration. Experimental ligand density is shown in grey surface and the ligands are shown in stick representation. Densities are shown at a contour level of 5.5 σ (panel C and D). Difference map densities are shown at a contour level of 8 σ (panel A and B) and 5 σ (panel C and D). The difference density disappears at 29 σ (panel A) and 27 σ (panel B).