Panel A shows a schematic representation of four C N B D subunits of an H C N channel in the apo state, where one subunit is subjected to directional perturbation at the ligand binding site to mimic a bound-like conformation. Panel B shows a comparison between predicted and experimentally observed displacements at the binding sites across different subunits, highlighting similarities or differences in the direction of structural changes. Panel C shows the variation in overlap values with increasing number of bound ligands, illustrating patterns of cooperative behavior across subunits along with corresponding binding trends. The x-axis represents the number of bound c A M P, and the y-axis represents the overlap value. The graph shows different binding patterns and cooperativity effects.
Analysis of cooperativity in ligand binding to HCN channels by LRT. (A) Principle of approach. Sketch of four CNBDS (I–IV) of HCN channel in apo state with empty cAMP-binding site (triangle). Forces (red arrows) applied to binding site in one subunit (I) mimic the binding site structure in cAMP-bound conformation (dotted line). (B) The forces for shifting ligand-binding site into holo-conformation (red arrows) are compared with the experimental structure displacements (green arrows) between apo- and holo-state of the binding sites from cryo-EM structures. Note that red and green arrows point in large in the same direction in subunit I and III (high positive overlap value) but into opposite directions in subunit II and IV (high negative overlap value). Hence, forces in monomer I reproduce its cAMP-bound state. The imposed holo-state in I also promotes conformational transition toward a cAMP-bound conformation in the opposite monomer III. This can be interpreted as positive cooperativity. Imposed binding in I in contrast disfavors ligand binding in lateral monomers II and IV, suggesting a negative cooperativity. (C) LRT-modeled sequential binding patterns of HCN1 (blue) and HCN4 (red) channels show in their overlap values the same pattern of positive and negative cooperativity as the experimentally derived Ka values for cAMP binding in HCN2 (black) by Kusch et al. (2012). Bottom row depicts in red the acutely perturbed subunit in the LRT analysis, proceeding perturbed subunits in blue, and unperturbed subunits in gray. Data in B and C reproduced with permission by Kunzmann et al. (2024). See caption Fig. 5 for the magnitude/lengths of the arrows.