Two aspartate residues in the distal C-helix make smaller 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 D631 and D634 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, 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 two-site independent binding model, which yielded values for affinity and energetics (ΔG, ΔH, and ΔS). (C) Bar graphs of high- and low-affinity binding values that arise from the fitting of the heat values with a two-site independent binding model for the binding of cGMP to the tetrameric HCN2 C-terminal wild-type protein and mutants containing single mutations, from the experiments shown in A. Values for binding affinity are from Table 1. (D) Bar graphs of values for thermodynamics that arise from the fitting of the heat values with a two-site independent binding model for the binding of cGMP to the wild-type and mutant HCN2 C-terminus. Values in C and D represent means ± SEM. Each mean was determined from independent ITC binding experiments.