(A) X-ray crystal structures of apo (this work) and (B) holo (PDB 3U10) conformations of the HCN2 CNBD colored according to protein secondary structure. Bound cAMP in the holo structure is shown as spheres. Helical domains are labeled D', E', A, B, C and P as was previously done for the holo structure (Zagotta et al., 2003). (C–D) Apo structure (red) overlaid with either the β-roll domain (C) or the α-helical termini (treated as a single domain) (D) of the holo structure (blue). The apo and holo structures superimpose with an RMSD of 0.49 Å over their β-roll domains, or 2.04 Å over their α-helical terminal domains (N-terminal helix excludes the initial D' segment). Hinge-like rotations that account for bulk of conformational changes between apo and holo structures: (E) N-terminal helical fragment (residues 508–534), (F) C-terminal helical fragment (residues 607–634). (G) Steric clashes between holo conformation of the C-terminus (blue) and apo conformation of the N-terminus (red). Residues that would clash are illustrated as spheres (M515, P516, L517 and L615, M621, A624, F625). (H) Cartoon illustrating the cAMP-induced rotation of the α-helical domains (cyan) about the rigid β-roll cage (magenta) that both caps the bound ligand (yellow) and places the N-terminal region in a favorable state for coordinating intersubunit interactions with neighboring CNBDs (indicated by a faded CNBD).