cAMP Modulation of the cytoplasmic domain in the HCN2 channel investigated by molecular simulations

Abstract

The hyperpolarization-activated cyclic nucleotide-modulated (HCN) cation channels are opened by membrane hyperpolarization, while their activation is modulated by the binding of cyclic adenosine monophosphate (cAMP) in the cytoplasm. Here we investigate the molecular basis of cAMP channel modulation by performing molecular dynamics simulations of a segment comprising the C-linker and the cyclic nucleotide binding domain (CNBD) in the presence and absence of cAMP, based on the available crystal structure of HCN2 from mouse. In presence of cAMP, the protein undergoes an oscillation of the quaternary structure on the order of 10 ns, not observed in the apoprotein. In contrast, the absence of ligand causes conformational rearrangements within the CNBDs, driving these domains to a more flexible state, similar to that described in CNBDs of other proteins. This increased flexibility causes a rather disordered movement of the CNBDs, resulting in an inhibitory effect on the channel. We propose that the cAMP-triggered large-scale oscillation plays an important role for the channel's function, being coupled to a motion of the C-linker which, in turn, modulates the gating of the channel.

FIGURE 1

(a) Structure of C-linker and CNBD of HCN2 from mouse (17): four subunits are arranged around the fourfold rotational symmetry axis, which is represented by the dashed line, forming the homotetramer. C-linkers are at the top and CNBDs at the bottom of the picture. Three subunits are drawn in cartoons and in the fourth, which is drawn in trace, secondary structure elements are colored in violet for helices and yellow for β-strands and one cAMP molecule is shown. (b) Upper view of the complex: stars indicate mass centers of opposite couples of adjacent subunits. (c) Diagram of one subunit of HCN2 C-linker and CNBD with cAMP in its binding site. (d) Cascade of helices rearrangements after ligand unbinding (20,23,28,34). (e) Representative snapshot of the apoprotein, to be compared with c: balls represent residues whose B-factor in the apoprotein is at least two (orange) and three (red) times that in the holoprotein, which are spread in the CNBD and E′- and F′-helices, including residues forming the binding site. (f) The most relevant motions in the holoprotein are oscillations of the quaternary structure, which are here identified by the distances between mass centers of opposite couples of adjacent subunits (stars as in b) and are plotted in Fig. 2, c and d. (g) B-factors in the holoprotein (black line) and in the apoprotein (red line) are plotted along the sequence. Secondary structure elements are indicated in violet for helices and yellow for β-strands, and are labeled.

FIGURE 2

Cα RMSD plots (a,b) and distances between mass centers of opposite couples of adjacent subunits (c,d) according to Fig. 1, b and f, in the holoprotein (a,c) and in the apoprotein (b,d). RMSD for the tetramer (solid) and average RMSD for single chains (shaded) are shown. For the holoprotein, distances and RMSD plots are correlated and display an oscillation that can be fitted by a sine function with a period of ∼13 ns and an amplitude of ∼0.03 nm for RMSD and ∼0.05 nm for both centers-of-mass distances.