Normal-Mode-Analysis-Guided Investigation of Crucial Intersubunit Contacts in the cAMP-Dependent Gating in HCN Channels

Authors

Farzana Marni, Virginia Commonwealth University
Shengjun Wu, Virginia Commonwealth University
Gaurav M. Shah, Virginia Commonwealth UniversityFollow
Xin-ping Xu, Virginia Commonwealth UniversityFollow
Amber R. Hackett, Virginia Commonwealth University
Changan Xie, Virginia Commonwealth UniversityFollow
Sabisha Shrestha, Virginia Commonwealth University
Lin Liu, Virginia Commonwealth University
Qinglian Liu, Virginia Commonwealth UniversityFollow
Lei Zhou, Virginia Commonwealth UniversityFollow

Document Type

Article

Original Publication Date

2012

Journal/Book/Conference Title

The Biophysical Journal

Volume

103

Issue

1

First Page

19

Last Page

28

DOI of Original Publication

10.1016/j.bpj.2012.05.030

Comments

Originally published at http://dx.doi.org/10.1016/j.bpj.2012.05.030

Under an Elsevier user license

Date of Submission

February 2015

Abstract

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.

Rights

From The Biophysical Journal, Marni, F., Wu, S., Shah, G.M., et al., Normal-Mode-Analysis-Guided Investigation of Crucial Intersubunit Contacts in the cAMP-Dependent Gating in HCN Channels, Vol. 103, Page 19, Copyright © 2012 Biophysical Society. Published by Elsevier Inc. Reprinted with permission.

Is Part Of

VCU Physiology and Biophysics Publications