title: Normal-mode-analysis-guided investigation of crucial intersubunit contacts in the cAMP-dependent gating in HCN channels.
abstract: Protein structures define a complex network of atomic interactions in three dimensions. Direct visualization of the structure and analysis of the interaction potential energy are not straightforward approaches to pinpoint the atomic contacts that are crucial for protein function. We used the tetrameric hyperpolarization-activated cAMP-regulated (HCN) channel as a model system to study the intersubunit contacts in cAMP-dependent gating. To obtain a systematic survey of the contacts between each pair of residues, we used normal-mode analysis, a computational approach for studying protein dynamics, and constructed the covariance matrix for C-α atoms. The significant contacts revealed by covariance analysis were further investigated by means of mutagenesis and functional assays. Among the mutant channels that show phenotypes different from those of the wild-type, we focused on two mutant channels that express opposite changes in cAMP-dependent gating. Subsequent biochemical assays on isolated C-terminal fragments, including the cAMP binding domain, revealed only minimal effects on cAMP binding, suggesting the necessity of interpreting the cAMP-dependent allosteric regulation at the whole-channel level. For this purpose, we applied the patch-clamp fluorometry technique and observed correlated changes in the dynamic, state-dependent cAMP binding in the mutant channels. This study not only provides further understanding of the intersubunit contacts in allosteric coupling in the HCN channel, it also illustrates an effective strategy for delineating important atomic contacts within a structure.
authors: Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, Virginia.
authors: Farzana
authors: F
authors: Marni
authors: Shengjun
authors: S
authors: Wu
authors: Gaurav M
authors: GM
authors: Shah
authors: Xin-ping
authors: XP
authors: Xu
authors: Amber R
authors: AR
authors: Hackett
authors: Changan
authors: C
authors: Xie
authors: Sabisha
authors: S
authors: Shrestha
authors: Lin
authors: L
authors: Liu
authors: Qinglian
authors: Q
authors: Liu
authors: Lei
authors: L
authors: Zhou
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D000242:Cyclic AMP / Q000737:chemistry*; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D051379:Mice; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D021122:Protein Subunits / Q000737:chemistry*; D014981:Xenopus
chemical_list: D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D021122:Protein Subunits; D000242:Cyclic AMP