Fig. 8: Trip8bnano binding to CNBD constructs with and without D and E helices.
From: A high affinity switch for cAMP in the HCN pacemaker channels
a Left, superposition of the unbound (PDB: 5U6O, blue) and cAMP-bound (PDB: 6UQF, gray) structures of HCN2 CNBD; these constructs do not include helices DE. Center, structure of HCN1 (PDB:6UQF) highlighting the salt bridge, conserved in HCN subtypes, between Asp629 (red sticks) on helix E and Arg593 (blue sticks) on helix C. Right, structural model of HCN2 CNBD (light blue) in complex with TRIP8bnano peptide (orange)33. Asp50 and 57 (red sticks) on the TRIP8bnano peptide form salt bridges with Arg 662 (blue sticks) of C-helix. b Examples of ITC thermogram obtained by titrating purified human HCN2 CNBD ΔC-term, ΔE and with TRIP8bnano peptide. Upper panel, heat changes (μcal/sec) during successive injections of TRIP8b peptide. Lower panel, binding curves obtained from data displayed in the upper panel. The peaks were integrated, normalized to TRIP8b peptide concentration and plotted against the molar ratio (TRIP8bnano/CNBD). Solid red line represents a nonlinear least-squares fit to a single-site binding model yielding, in the present examples, equilibrium dissociation constant (KD) and stoichiometry (N) values as shown. Mean KD,N values and statistical analysis are reported in Supplementary Table 3. c Dissociation constant (KD) values ± SEM of CNBD ΔC-term, ΔE, ΔDE’ and ΔDE. d Mean activation curves of HCN4 FL (black) and ΔE (green) in control solution (full circles), with 15 µM and 60 µM cAMP for FL and ΔE respectively (empty circles), and with cAMP + 1 µM of purified TRIP8bnano in the patch pipette (diamonds). Lines show Boltzmann fitting to the data. Half-activation voltage (V1/2) and inverse slope factor (k) values are reported in Supplementary Table 4 together with the details on statistical analysis. Data are presented as mean ± SEM (where not visible, the error bars are within the symbol). e Mean shift due to TRIP8bnano ± SEM calculated from data in (d).
