Fig. 3: Analyses of MD simulations of the wild-type and K464E mHCN2 channel.

a Average occurrence frequency of hydrogen bond interactions between two opposite subunits involving K464. K464 resides on subunit i, and the interaction partners M150, E243, D244, and E247 reside on subunit i + 2. As to M155, we only considered the backbone oxygen as H-bond acceptor; for E243, D244, and E247, we only considered the side-chain oxygen atoms, as we considered these interactions more favorable compared to backbone interactions. b Average rotation angle relative to the channel pore in the starting structure. The direction of rotation is visualized by the scheme below the panels. c Overlay of CL-CNBDs after superimposing the pore regions. From left to right, the panels show the overlay of the average apo wild-type mHCN2 (dark blue) and the average K464E mHCN2 (green) or the average cAMP-bound wild-type mHCN2 (orange) from MD simulations, the overlay of the apo (light blue, PDB ID 5U6O⁶) and cAMP-bound (yellow, PDB ID 5U6P⁶) cryo-EM structures of hHCN1, and the overlay of the apo (magenta, PDB ID 6GYN¹⁸ and cAMP-bound (dark cyan, PDB ID 6GYO⁶¹ cryo-EM structures of hHCN4. Helices are shown as cylinders. The arrows indicate the direction of rotation relative to the respective apo structures. The labels depict the angle of rotation relative to the respective apo structures. A′- and B′-labels denote the first two helices of the CL. In a and b, the average values (black lines) were calculated individually for each of the four subunits and throughout 20 independent MD simulation replicas (n = 80) with the individual data points shown as circles. The boxes denote the range from the 25th to 75th percentile and include 50% of the data points. The whiskers denote the 5th to 95th percentile and include 90% of the data points (p-value by t-test; *p < 0.05; **p < 0.01; n.s. not significantly different).