Fig. 4: Analysis of the salt bridge interaction between the E-helix and the C-helix.

a Ribbon representation of the D and E helices, in yellow, together with the C-helix of the CNBD, in gray (PDB:6UQF). The residues forming the salt bridge interaction (dotted line) are shown as sticks and labeled. Numbering refers to hHCN2 (H2) and rbHCN4 (H4) sequences. b Mean activation curves, measured by patch clamp, of HCN4 D750 (purple) in control solution (full symbol, solid line) and with 5 µM cAMP in the pipette solution (empty symbol, broken line). Activation curves of wild type HCN4 are plotted in black, without symbols, for comparison (replotted from FL in Fig. 2b). Half-activation voltage values (V_1/2) and inverse slope factors (k) are reported in Supplementary Table 2 together with the details on statistical analysis. Data are shown as mean ± SEM. c Shift of V_1/2 as a function of cAMP concentration for HCN4 D750A construct (purple symbols). Each data point is an average of n ≥ 3 experiments (exact numbers are reported in the source data file). Data fit to the Hill equation (solid line) yielded half maximal effective concentration (K_1/2) of 10.4 µM and Hill coefficient (nH) of 0.8. Data are presented as mean ± SEM. Data for HCN4 FL (black line) and ΔE (green line) are replotted form Fig. 2c. d Representative ITC measurements of cAMP binding to purified hHCN2 CNBD D698A and R662A. Top panels show heat changes (μcal/sec) after cAMP injection into the chamber containing CNBD. Bottom panels show binding curves obtained from data displayed in the upper panel. The peaks were integrated, normalized to cAMP concentration, and plotted against the molar ratio (cAMP/CNBD). Solid red line represents a nonlinear least-squares fit to a single-site binding model yielding, in the present examples, equilibrium dissociation constant (K_D) and stoichiometry (N) values as shown. Mean K_D and N values are reported in Supplementary Table 3 together with the statistical analysis. e Calculated K_D values ± SEM for CNBD D698A and R662A (empty purple bars). Values for CNBD ΔC-term and ΔE are replotted as black and green bars without data points, from Fig. 2e.