Two residues in the PBC and three other residues in the C-helix make strong contributions to cGMP binding to the tetrameric HCN2 C-terminus. (A) A ribbon diagram showing the wild-type HCN2 C-linker and CNBD bound to cGMP. The positions of R591 and T592, in the PBC, and L633A and R635, in the distal part of the C-helix, are shown. Residues distal to R635 (including K638) were not resolved in this structure. PDB accession no. 1Q3E. (B) Plots of heat produced upon progressive injections of cGMP to 200 μM HCN2 C-terminus wild-type or mutant proteins as measured by ITC. The inflections in the top plot arise from injections of cGMP, where each inverted peak shows the heat difference between the sample and reference compartment. The peaks decrease in magnitude as binding sites become saturated. The lower plot shows values determined by integration of the area under the peaks from the upper plot versus the ratio of injected ligand to protein. The solid line through the values represents a single binding site model, which yielded values for affinity and energetics (ΔG, ΔH, and ΔS). (C) A bar plot of values for the single affinity binding of cGMP to the tetrameric HCN2 C-terminal wild-type protein and mutants containing single mutations, from the experiments shown in A. Values represent means ± SEM. Each mean was determined from independent ITC binding experiments. Values for binding affinity are also shown in Table 1.