Fig 1.
Detecting cNMP binding to the HCN2 C-linker/CNBDs with SPR.
(A) Cartoon of one of the four subunits of HCN channels. The transmembrane segments S1-S6 are grey, the C-linker is red, the CNBD domain is blue and the C-helix is green. cAMP bound to the CNBD and c-diGMP bound at the proposed site formed by the C-linker are depicted by yellow and cyan ellipses. (B) Cartoon of the mHCN2 C-linker/CNBD immobilized on the NTA chip surface using Ni2+-NTA and the N-terminal 6-His tag coupling. In the ribbon representation of the C-linker/CNBD [15], the C-linker is red, the β-roll and helices A, P and B are blue, and the C-helix is green. cAMP bound inside the β-roll cavity is yellow. (C) and (D) Representative SPR sensorgrams recorded for the immobilized HCN2 C-linker/CNBDs with the indicated concentrations of cAMP (C) and cGMP (D). C-linker/CNBDs immobilized on the same surface were used in (C) and (D). (E) Plots of the SPR response at 30 s after the start of the injection versus total cAMP (filled circles) and cGMP (open circles) concentration for sensorgrams shown in (C) and (D). The lines represent fits of the data with Hill equation. The binding affinities were 2.5 ± 0.4 μM for cAMP and 12.3 ± 1.1 μM for cGMP.
Table 1.
Cyclic nucleotide binding affinities for the C-linker/CNBDs of HCN channels determined with SPR.
Fig 2.
L586W mutation decreases cyclic nucleotide affinity to the C-linker/CNBDs of HCN2 channels.
(A) Enlarged view of the cAMP binding pocket and the Trp residue introduced at position 586. The ribbon representation for mHCN2-L586W was obtained with SWISS-MODEL [57] based on the crystal structure of the mHCN2 C-linker/CNBD [15]. cAMP is colored in yellow. (B) and (C) Representative SPR sensorgrams recorded for the immobilized HCN2-L586W C-linker/CNBDs with the indicated concentrations of cAMP (B) and cGMP (C). C-linker/CNBDs immobilized on the same surface were used in (B) and (C). (D) Plots of the SPR response at 30 s after the start of the injection versus total cAMP (filled squares) and cGMP (open squares) concentration for sensorgrams shown in (B) and (C). The lines represent fits of the data with Hill equation. The binding affinities were 10.6 ± 1.5 μM for cAMP and ≥70.1 ± 3.8 μM for cGMP.
Fig 3.
Detecting cNMP binding to the HCN4 C-linker/CNBDs with SPR.
(A) and (B) Representative SPR sensorgrams recorded for the immobilized hHCN4 C-linker/CNBDs with the indicated concentrations of cAMP (A) and cGMP (B). C-linker/CNBDs immobilized on the same surface were used in (A) and (B). (C) Plots of the SPR response at 30 s after the start of the injection versus total cAMP (filled diamonds) and cGMP (open diamonds) concentration for sensorgrams shown in (A) and (B). The lines represent fits of the data with Hill equation. The binding affinities were 1.4 ± 0.3 μM for cAMP and 5.6 ± 0.2 μM for cGMP.
Fig 4.
Cyclic dinucleotides do not bind to the HCN4 and HCN2 C-linker/CNBDs.
(A) Cartoon of the hHCN4 C-linker/CNBD immobilized on the NTA chip surface using Ni2+-NTA and the N-terminal 6-His tag coupling. The same color coding as in Fig 1B. c-di-GMP placed in the proposed CLP site is shown in cyan. The ribbon representation of the C-linker/CNBD is according to ref [14]. (B) Representative SPR sensorgrams recorded for the immobilized hHCN4 C-linker/CNBDs in the presence of 300 μM c-di-GMP (green), 300 μM c-di-GMP and 100 μM cAMP (grey), and 300 μM cGMP (red). All analytes were injected over the same surface with immobilized hHCN4 C-linker/CNBDs. No increase in the binding response was detected upon injection of c-di-GMP in the presence or absence of cAMP (grey and green traces). (C) Representative SPR sensorgrams recorded for the immobilized hHCN4 C-linker/CNBDs in the presence of 300 μM c-di-AMP (green), 300 μM c-di-AMP and 100 μM cAMP (grey), and 300 μM cAMP (red). No increase in the binding response was detected upon injection of c-di-AMP in the presence or absence of cAMP (grey and green traces). All analytes were injected over the same surface with immobilized hHCN4 C-linker/CNBDs. (D) Representative SPR sensorgrams recorded for the immobilized mHCN2 C-linker/CNBDs in the presence of 300 μM c-di-AMP (green), 300 μM c-di-AMP and 100 μM cAMP (grey), and 300 μM cAMP (red). No increase in the binding response was detected upon injection of c-di-AMP in the presence or absence of cAMP (grey and green traces). All analytes were injected over the same surface with immobilized mHCN2 C-linker/CNBDs.
Fig 5.
Cyclic nucleotide and cyclic dinucleotide binding to the monomeric and tetrameric HCN4 C-linker/CNBD tested with ITC.
Thermograms of successive injections of 1.25 μl of cAMP (A and D), c-di-GMP (B and E) and c-di-GMP in the presence of cAMP (C and F) (top panels) and the corresponding binding isotherms (bottom panels). For experiments in (C) 100 μM cAMP and in (F) 1 mM cAMP was present in both the protein and ligand solutions. Monomeric 6-His tagged hHCN4 C-linker/CNBDs at 6 μM concentration, purified in the same manner as for the SPR-based experiments, were used for experiments in (A-C). hHCN4 C-linker/CNBDs after the MBP tag cleavage at 80 μM concentration were used for experiments in (D-F). The binding isotherms were obtained by integrating the peaks in the top panels, normalizing the obtained values by the cAMP concentration and plotting them against the molar ratio of cAMP to the protein. The lines represent a nonlinear least-square fit to a single–site binding model for (A-C) and a two independent binding site model for (D-F). The binding affinities for cAMP were 1.1 ± 0.5 μM in (A), and 1.7 ± 0.3 μM and 0.03 ± 0.02 μM in (D).
Fig 6.
Structural model of the c-di-GMP regulation.
(A) Ribbon representation of transmembrane segments of two opposing subunits and the C-linker/CNBDs of the two subunits adjacent to them from the full-length structure of HCN1 channels [45]. The transmembrane segments of the subunits containing the C-linker/CNBDs shown in the figure and the C-linker/CNBDs of the subunits containing transmembrane segments shown in the figure are omitted for clarity. In the crystal structure C-linkers are making direct contacts with S4-S5 linkers and HCN domains from only the adjacent subunits. Transmembrane segments (TM) are shown in grey, S4-S5 linkers and HCN domains are orange, C-linkers are red, β-roll and helices A, P and B are blue, and the C-helix and the distal C-terminus are green. cAMP bound inside the β-roll cavity is yellow. c-di-GMP placed in the proposed CLP site is cyan. (B) Structural alignment of the C-linker/CNBD from the full-length HCN1 structure [45] shown in gray and isolated C-linker/CNBD of HCN2 channels [15] shown in magenta.