Investigation of Cyclic AMP Binding Interactions with Isolated Cyclic Nucleotide Binding Domain of HCN1 Channel using Atomistic Molecular Dynamics Simulations at Microsecond Timescale

Abstract

Hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) is a homotetrameric, voltage-gated potassium/sodium channel whose activity is modulated by cyclic AMP. The binding of cAMP to an intracellular cyclic nucleotide binding domain (CNBD) promotes an increase in channel activity through a depolarizing shift in the voltage threshold necessary for channel activation, as well as kinetically favoring the active channel conformation, leading to improved conductance. While the precise mechanisms of this change are still being investigated, the shift in the CNDB region, which is induced by the binding of cAMP, is able to be propagated to the remainder of the channel through an adjacent C-linker, causing an alteration in the channel that leads to increased conductance. Although static structures of both the apo and holo channel states have been solved and compared, there is still a need to understand the precise binding interactions which occur between the cyclic nucleotide and the binding pocket, as well as the dynamic changes to the local protein structure that might lead to the aforementioned changes in activity. Using atomistic molecular dynamics simulations, we have simulated both the apo and holo forms of the isolated CNBD region at a microsecond timescale, and have performed a variety of analyses to relate the two conformations and investigate the interactions of the ligand with the binding pocket. We have also used enhanced sampling techniques to calculate the binding free energy of cAMP. This study provides a computational framework for the study of HCN1-ligand interactions.