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Structure of KCNH2 cyclic nucleotide-binding homology domain reveals a functionally vital salt-bridge
Human KCNH2 channels (hKCNH2, ether-à-go-go [EAG]–related gene, hERG) are best known for their contribution to cardiac action potential repolarization and have key roles in various pathologies. Like other KCNH family members, hKCNH2 channels contain a unique intracellular complex, consisting of an N...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Rockefeller University Press
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141593/ https://www.ncbi.nlm.nih.gov/pubmed/32191791 http://dx.doi.org/10.1085/jgp.201912505 |
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author | Ben-Bassat, Ariel Giladi, Moshe Haitin, Yoni |
author_facet | Ben-Bassat, Ariel Giladi, Moshe Haitin, Yoni |
author_sort | Ben-Bassat, Ariel |
collection | PubMed |
description | Human KCNH2 channels (hKCNH2, ether-à-go-go [EAG]–related gene, hERG) are best known for their contribution to cardiac action potential repolarization and have key roles in various pathologies. Like other KCNH family members, hKCNH2 channels contain a unique intracellular complex, consisting of an N-terminal eag domain and a C-terminal cyclic nucleotide-binding homology domain (CNBHD), which is crucial for channel function. Previous studies demonstrated that the CNBHD is occupied by an intrinsic ligand motif, in a self-liganded conformation, providing a structural mechanism for the lack of KCNH channel regulation by cyclic nucleotides. While there have been significant advancements in the structural and functional characterization of the CNBHD of KCNH channels, a high-resolution structure of the hKCNH2 intracellular complex has been missing. Here, we report the 1.5 Å resolution structure of the hKCNH2 channel CNBHD. The structure reveals the canonical fold shared by other KCNH family members, where the spatial organization of the intrinsic ligand is preserved within the β-roll region. Moreover, measurements of small-angle x-ray scattering profile in solution, as well as comparison with a recent NMR analysis of hKCNH2, revealed high agreement with the crystallographic structure, indicating an overall low flexibility in solution. Importantly, we identified a novel salt-bridge (E807-R863) which was not previously resolved in the NMR and cryo-EM structures. Electrophysiological analysis of charge-reversal mutations revealed the bridge’s crucial role in hKCNH2 function. Moreover, comparison with other KCNH members revealed the structural conservation of this salt-bridge, consistent with its functional significance. Together with the available structure of the mouse KCNH1 intracellular complex and previous electrophysiological and spectroscopic studies of KCNH family members, we propose that this salt-bridge serves as a strategically positioned linchpin to support both the spatial organization of the intrinsic ligand and the maintenance of the intracellular complex interface. |
format | Online Article Text |
id | pubmed-7141593 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-71415932020-10-06 Structure of KCNH2 cyclic nucleotide-binding homology domain reveals a functionally vital salt-bridge Ben-Bassat, Ariel Giladi, Moshe Haitin, Yoni J Gen Physiol Communication Human KCNH2 channels (hKCNH2, ether-à-go-go [EAG]–related gene, hERG) are best known for their contribution to cardiac action potential repolarization and have key roles in various pathologies. Like other KCNH family members, hKCNH2 channels contain a unique intracellular complex, consisting of an N-terminal eag domain and a C-terminal cyclic nucleotide-binding homology domain (CNBHD), which is crucial for channel function. Previous studies demonstrated that the CNBHD is occupied by an intrinsic ligand motif, in a self-liganded conformation, providing a structural mechanism for the lack of KCNH channel regulation by cyclic nucleotides. While there have been significant advancements in the structural and functional characterization of the CNBHD of KCNH channels, a high-resolution structure of the hKCNH2 intracellular complex has been missing. Here, we report the 1.5 Å resolution structure of the hKCNH2 channel CNBHD. The structure reveals the canonical fold shared by other KCNH family members, where the spatial organization of the intrinsic ligand is preserved within the β-roll region. Moreover, measurements of small-angle x-ray scattering profile in solution, as well as comparison with a recent NMR analysis of hKCNH2, revealed high agreement with the crystallographic structure, indicating an overall low flexibility in solution. Importantly, we identified a novel salt-bridge (E807-R863) which was not previously resolved in the NMR and cryo-EM structures. Electrophysiological analysis of charge-reversal mutations revealed the bridge’s crucial role in hKCNH2 function. Moreover, comparison with other KCNH members revealed the structural conservation of this salt-bridge, consistent with its functional significance. Together with the available structure of the mouse KCNH1 intracellular complex and previous electrophysiological and spectroscopic studies of KCNH family members, we propose that this salt-bridge serves as a strategically positioned linchpin to support both the spatial organization of the intrinsic ligand and the maintenance of the intracellular complex interface. Rockefeller University Press 2020-03-19 /pmc/articles/PMC7141593/ /pubmed/32191791 http://dx.doi.org/10.1085/jgp.201912505 Text en © 2020 Ben-Bassat et al. http://www.rupress.org/terms/https://creativecommons.org/licenses/by-nc-sa/4.0/This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/). |
spellingShingle | Communication Ben-Bassat, Ariel Giladi, Moshe Haitin, Yoni Structure of KCNH2 cyclic nucleotide-binding homology domain reveals a functionally vital salt-bridge |
title | Structure of KCNH2 cyclic nucleotide-binding homology domain reveals a functionally vital salt-bridge |
title_full | Structure of KCNH2 cyclic nucleotide-binding homology domain reveals a functionally vital salt-bridge |
title_fullStr | Structure of KCNH2 cyclic nucleotide-binding homology domain reveals a functionally vital salt-bridge |
title_full_unstemmed | Structure of KCNH2 cyclic nucleotide-binding homology domain reveals a functionally vital salt-bridge |
title_short | Structure of KCNH2 cyclic nucleotide-binding homology domain reveals a functionally vital salt-bridge |
title_sort | structure of kcnh2 cyclic nucleotide-binding homology domain reveals a functionally vital salt-bridge |
topic | Communication |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141593/ https://www.ncbi.nlm.nih.gov/pubmed/32191791 http://dx.doi.org/10.1085/jgp.201912505 |
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