Dynamic rearrangement of the intrinsic ligand regulates KCNH potassium channels

Dai, Gucan; James, Zachary M.; Zagotta, William N.

doi:10.1085/jgp.201711989

Cited by 33 publications

(55 citation statements)

References 51 publications

(116 reference statements)

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“…Thus, it appears that the saltbridge serves as a linchpin, limiting the conformational landscape of the ILM, while allowing interaction of the post-CNBHD with the eag domain in mouse KCNH1 channels. This can support recent results demonstrating that the ILM undergoes a defined conformational change during voltage-dependent channel potentiation, rather than dissociation from its binding pocket upon the relative rearrangement between the eag domain and the CNHBD (Dai et al, 2018).…”

Section: Discussionsupporting

confidence: 90%

Structure of KCNH2 cyclic nucleotide-binding homology domain reveals a functionally vital salt-bridge

Ben-Bassat

Giladi

Haitin

2019

Preprint

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Human KCNH2 are key channels governing cardiac repolarization. Here, a 1.5 Å resolution structure of their cyclic nucleotide-binding homology domain is presented. Structural analysis and electrophysiological validation reveal a novel salt-bridge, playing an important role in hKCNH2 functional regulation.3 AbstractHuman KCNH2 (hKCNH2, Ether-à-go-go (EAG)-Related Gene, hERG) are best known for their role in cardiac action potentials repolarization and have key roles in various pathologies. As other KCNH family members, hKCNH2 contains a unique intracellular complex crucial for channel function, consisting of an N-terminal eag domain and a C-terminal cyclic nucleotide-binding homology domain (CNBHD). Previous studies demonstrated that the CNBHD is occupied by an intrinsic ligand motif (ILM), in a self-liganded conformation, providing a structural mechanism for the lack of KCNH channels regulation by cyclic nucleotides. While significant advancements in structural and functional characterizations of the CNBHD of KCNH channels have been made, a high-resolution structure of the hKCNH2 intracellular complex was 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 ILM is preserved within the b-roll region. Moreover, measurements of small-angle X-ray scattering profile in solution, as well as comparison with a recent nuclear magnetic resonance (NMR) analysis of hKCNH2, revealed high agreement with the 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 cryogenic electron microscopy (cryo-EM) structures. Strikingly, electrophysiological analysis of charge reversal mutations revealed its crucial role for 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 ILM and the maintenance of the intracellular complex interface.

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Section: Discussionsupporting

confidence: 90%

Structure of KCNH2 cyclic nucleotide-binding homology domain reveals a functionally vital salt-bridge

Ben-Bassat

Giladi

Haitin

2019

Preprint

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“…However, most of these studies focused on state-dependent changes in fluorescence due to the environmentally-sensitive emission of L-Anap (21,24,25,(34)(35)(36)(37). L-Anap has also been recently used as a FRET donor in studies to discern movement in ion channels between domains or between the channel and the membrane (17,22,24,25). Although these studies demonstrated the potential power of using L-Anap as a donor for tmFRET, the distances and distance changes measured were unexpected and difficult to interpret, given the limited structures available.…”

Section: Discussionmentioning

confidence: 99%

“…L-Anap's small size, short linker, and spectral properties makes it useful for tmFRET experiments (17,22,24,25). The peak absorption wavelength of L-Anap is 360 nm (16).…”

Section: Specific Labeling With Fluorophore and Transition Metal Ionmentioning

confidence: 99%

Visualizing conformational dynamics of proteins in solution and at the cell membrane

Gordon

Munari

Zagotta

2018

Preprint

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Conformational dynamics underlie enzyme function, yet are generally inaccessible via traditional structural approaches. FRET has the potential to measure conformational dynamics in vitro and in intact cells, but technical barriers have thus far limited its accuracy, particularly in membrane proteins. Here, we combine amber codon suppression to introduce a donor fluorescent noncanonical amino acid with a new, biocompatible approach for labeling proteins with acceptor transition metals in a method called ACCuRET (Anap Cyclen-Cu 2+ resonance energy transfer). We show that ACCuRET measures absolute distances and distance changes with high precision and accuracy using maltose binding protein as a benchmark. Using cell unroofing, we show that ACCuRET can accurately measure rearrangements of proteins in native membranes. Finally, we implement a computational method for correcting the measured distances for the distance distributions observed in proteins. ACCuRET thus provides a flexible, powerful method for measuring conformational dynamics in both soluble proteins and membrane proteins.

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“…; Dai et al . ). However, achieving further understanding of the mechanisms involved in VSP activity through the use of fluorescent unnatural amino acids will probably require overcoming several important issues.…”

Section: Future Directionsmentioning

confidence: 97%

“…Our recent application of the Anap method to Ci-VSP has proven to be a powerful tool for detecting stimulus-induced motion of the cytoplasmic region, which has been difficult to study using conventional methods, such as approaches based on cysteine-maleimide reactions. Anap has also been utilized as a reporter of structural changes in other membrane proteins (Zagotta et al 2016;Soh et al 2017;Dai et al 2018). However, achieving further understanding of the mechanisms involved in VSP activity through the use of fluorescent unnatural amino acids will probably require overcoming several important issues.…”

Section: Future Directionsmentioning

confidence: 99%

Dynamic structural rearrangements and functional regulation of voltage‐sensing phosphatase

Sakata

Okamura

2018

The Journal of Physiology

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The voltage‐sensing phosphatase (VSP) consists of a voltage sensor domain (VSD) and a cytoplasmic catalytic region. The latter contains a phosphatase domain and a C2 domain, showing remarkable similarity to the tumour suppressor enzyme PTEN. In VSP, membrane depolarization induces a conformational change in the VSD, which activates the phosphoinositide phosphatase. The final outcome in VSP is enzymatic activity in the cytoplasmic region, unlike in voltage‐gated ion channels where conformational change of the transmembrane pore is induced by the VSD. Therefore, it is crucial to detect structural change in the cytoplasmic catalytic region to gain insights into the operating mechanisms of VSP. This review summarizes a recent study in which a method of genetic incorporation of a non‐canonical amino acid, Anap, was used to detect dynamic membrane voltage‐controlled rearrangements of the structure of the catalytic region of sea squirt VSP (Ci‐VSP). Upon membrane depolarization, both the phosphatase domain and the C2 domain move in a similar time frame, suggesting that the two regions are coupled to each other. Measurement of Förster resonance energy transfer (FRET) between Anap introduced into the C2 domain of Ci‐VSP and dipicrylamine in the cell membrane suggested no large movement of the enzyme towards the membrane. Fluorescence changes in Anap induced by different membrane potentials indicate the presence of multiple conformations of the active enzyme.

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Dynamic rearrangement of the intrinsic ligand regulates KCNH potassium channels

Cited by 33 publications

References 51 publications

Structure of KCNH2 cyclic nucleotide-binding homology domain reveals a functionally vital salt-bridge

Structure of KCNH2 cyclic nucleotide-binding homology domain reveals a functionally vital salt-bridge

Visualizing conformational dynamics of proteins in solution and at the cell membrane

Dynamic structural rearrangements and functional regulation of voltage‐sensing phosphatase

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