Cyclic Nucleotide-binding Homology Domain Research Articles

An intracellular hydrophobic nexus critical for hERG1 channel slow deactivation

Slow deactivation is a critical property of voltage-gated K+ channels encoded by the human Ether-à-go-go-Related Gene 1 (hERG). hERG1 channel deactivation is modulated by interactions between intracellular N-terminal Per-Arnt-Sim (PAS) and C-terminal cyclic nucleotide-binding homology (CNBh) domains. The PAS domain is multipartite, comprising a globular domain (gPAS; residues 26–135) and an N-terminal PAS-cap that is further subdivided into an initial unstructured “tip” (residues 1–12) and an amphipathic α-helical region (residues 13–25). Although the PAS-cap tip has long been considered the effector of slow deactivation, how its position near the gating machinery is controlled has not been elucidated. Here, we show that a triad of hydrophobic interactions among the gPAS, PAS-cap α helix, and the CNBh domains is required to support slow deactivation in hERG1. The primary sequence of this “hydrophobic nexus” is highly conserved among mammalian ERG channels but shows key differences to fast-deactivating Ether-à-go-go 1 (EAG1) channels. Combining sequence analysis, structure-directed mutagenesis, electrophysiology, and molecular dynamics simulations, we demonstrate that polar serine substitutions uncover an intermediate deactivation mode that is also mimicked by deletion of the PAS-cap α helix. Molecular dynamics simulation analyses of the serine-substituted channels show an increase in distance among the residues of the hydrophobic nexus, a rotation of the intracellular gating ring, and a retraction of the PAS-cap tip from its receptor site near the voltage sensor domain and channel gate. These findings provide compelling evidence that the hydrophobic nexus coordinates the respective components of the intracellular gating ring and positions the PAS-cap tip to control hERG1 deactivation gating.

New insights into the structure and function of CNNM proteins.

Magnesium (Mg2+ ) is the most abundant divalent cation in cells and plays key roles in almost all biological processes. CBS-pair domain divalent metal cation transport mediators (CNNMs) are a newly characterized class of Mg2+ transporters present throughout biology. Originally discovered in bacteria, there are four CNNM proteins in humans, which are involved in divalent cation transport, genetic diseases, and cancer. Eukaryotic CNNMs are composed of four domains: an extracellular domain, a transmembrane domain, a cystathionine-β-synthase (CBS)-pair domain, and a cyclic nucleotide-binding homology domain. The transmembrane and CBS-pair core are the defining features of CNNM proteins with over 20 000 protein sequences known from over 8000 species. Here, we review the structural and functional studies of eukaryotic and prokaryotic CNNMs that underlie our understanding of their regulation and mechanism of ion transport. Recent structures of prokaryotic CNNMs confirm the transmembrane domain mediates ion transport with the CBS-pair domain likely playing a regulatory role through binding divalent cations. Studies of mammalian CNNMs have identified new binding partners. These advances are driving progress in understanding this deeply conserved and widespread family of ion transporters.

The hERG PAS domain: A major regulator of IKr and a promising anti-arrhythmic drug target.

The hERG1 potassium channel conducts the critical cardiac repolarizing current IKr. Given its role in mediating electrical excitability, several groups have investigated the antiarrhythmic capacity of hERG1 activators in cardiac diseases marked by prolonged action potential duration (APD), such as long QT Syndrome. hERG1 activators have had limited success due to their off-target effects. We recently generated a single chain variable fragment (scFv) antibody, scFv2.10, that selectively binds the hERG1 Per-Arnt-Sim (PAS) domain. The PAS domain binds to the C-terminal cyclic nucleotide binding homology domain (CNBHD) and slows channel activation and promotes inactivation. scFv2.10 disrupts the PAS-CNBHD interaction, which in turn causes a two-fold increase in the time course of hERG1 activation and inactivation recovery, thereby increasing hERG1 current in HEK293 cells. In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), disrupting the PAS-CNBHD interaction with scFv2.10 increases IKr and shortens APD. We hypothesized that the hERG1 PAS domain could represent a novel therapeutic target for diseases of impaired cardiac repolarization. To test this, we measured the antiarrhythmic capacity of scFv2.10 on a novel hiPSC-CM line derived from a patient with Jervell and Lange Nielsen syndrome (JLN). JLN syndrome is a severe form of long QT Syndrome Type 1 characterized by increased APD and incidence of sudden cardiac death. Compared to hiPSC-CMs derived from a healthy genetic background, JLN hiPSC-CMs display hallmarks of pro-arrhythmia including prolonged APD, increased action potential variability, and early afterdepolarizations. scFv2.10 reduced APD, action potential variability, and the incidence of early afterdepolarizations in JLN hiPSC-CMs, compared to GFP controls. These data demonstrate that the hERG1 PAS domain is a potential therapeutic target to treat disorders of electrical excitability.

Using the photo-crosslinkable non-canonical amino acid BZF to capture U.V.-driven, state-dependent disruption of kinetics and voltage dependence of activation in hERG potassium channels.

Human ether-à-go-go related gene (hERG) voltage-activated potassium channels are critical for cardiac excitability. hERG deactivation is regulated by interaction between the N-terminal Per-Arnt-Sim (PAS) domain and the C-terminal cyclic nucleotide binding homology domain (CNBHD). Now, we aim to understand the how the conformational dynamics and accessible landscape of the hERG PAS domain contributes to channel kinetics and voltage dependence. To achieve this, we utilized the non-canonical amino acid 4-Benzoyl-L-phenylalanine (BZF) which is a photo-activatable cross-linkable probe, that when irradiated with ultraviolet (U.V.) light forms a covalent cross-link with C-H bond-containing groups, enabling selective and potent U.V.-driven state-dependent photoinactivation modification of ion channel dynamics. This is because BZF can form bonds to nearby atoms via cross-linkable reactivity reducing degrees of freedom within a domain of the channel. Here we incorporate BZF directly into the hERG potassium channel using TAG codon suppression that, upon U.V exposure, shows a large change in the biophysical properties of the channel when irradiated at −100 mV when the channels are closed. When compared to wild-type hERG1a, hERG1a-BZF shows a U.V. dose-dependent change (speeding up) of channel deactivation. Additionally, hERG1a-BZF exhibits a marked change (right-shift) in the voltage-dependence of activation when irradiated at −100 mV when the channels are closed. These effects are not observed when the hERG1a-BZF channel is irradiated in at 0 mV when the channels are open. This approach demonstrates that direct photo-crosslinking of KCNH hERG channels causes a measurable change in biophysical parameters and this effect is state-dependent (occurring here in the closed state but not the open state). We propose that altered channel gating is as a direct result of reduced dynamic motions in the hERG channel due to photo-chemical crosslinking.

A possible role for phase separation in heteromeric biogenesis of hERG1a/1b channels.

The human ether-a-go-go related gene (hERG) encodes a K+ channel important for repolarizing the cardiac action potential. Cardiac IKr is produced by heteromeric assemblies of hERG1a and 1b subunits, and depletion of hERG1b subunits confers proarrhythmic behavior in cellular cardiac models. hERG1a homomers can traffic to the membrane, but hERG1b homomers fail to progress efficiently out of the endoplasmic reticulum (ER). We found that hERG1b protein is sequestered and forms punctate intracellular structures when expressed heterologously in HeLa cells. When co-expressed with hERG1a, previously shown to promote hERG1b maturation, few puncta are observed and both signals are robustly present at the plasma membrane. We show that 1b puncta are primarily localized to the ER space and do not overlap with other membranous compartments, including COPII, lysosome, and autophagosome. Testing the hypothesis that hERG1b puncta are condensates arising from liquid-liquid phase separation, we used live cell imaging and observed that sequestered hERG1b puncta undergo fusion events, although rarely as might be expected from ER-delimited membrane proteins. Using truncation experiments, we found that removing the disordered C-terminus altered the size and distribution of the puncta but removing the structured cyclic nucleotide-binding homology domain (CNBhD) abolished the puncta entirely. These data suggest the puncta are formed by multivalent interactions of ordered and disordered domains of hERG1b and may sequester hERG1b in the absence of hERG1a. In this way sequestration in puncta may limit deleterious homomeric expression of hERG1b and thus promote heteromeric hERG1a/1b channel assembly.

14-3-3 proteins regulate cullin 7-mediated Eag1 degradation

BackgroundMutations in the human gene encoding the neuron-specific Eag1 (KV10.1; KCNH1) potassium channel are linked to congenital neurodevelopmental diseases. Disease-causing mutant Eag1 channels manifest aberrant gating function and defective protein homeostasis. Both the E3 ubiquitin ligase cullin 7 (Cul7) and the small acid protein 14-3-3 serve as binding partners of Eag1. Cul7 mediates proteasomal and lysosomal degradation of Eag1 protein, whereas over-expression of 14-3-3 notably reduces Eag1 channel activity. It remains unclear whether 14-3-3 may also contribute to Eag1 protein homeostasis.ResultsIn human cell line and native rat neurons, disruptions of endogenous 14-3-3 function with the peptide inhibitor difopein or specific RNA interference up-regulated Eag1 protein level in a transcription-independent manner. Difopein hindered Eag1 protein ubiquitination at the endoplasmic reticulum and the plasma membrane, effectively promoting the stability of both immature and mature Eag1 proteins. Suppression of endogenous 14-3-3 function also reduced excitotoxicity-associated Eag1 degradation in neurons. Difopein diminished Cul7-mediated Eag1 degradation, and Cul7 knock-down abolished the effect of difopein on Eag1. Inhibition of endogenous 14-3-3 function substantially perturbed the interaction of Eag1 with Cul7. Further structural analyses suggested that the intracellular Per-Arnt-Sim (PAS) domain and cyclic nucleotide-binding homology domain (CNBHD) of Eag1 are essential for the regulatory effect of 14-3-3 proteins. Significantly, suppression of endogenous 14-3-3 function reduced Cul7-mediated degradation of disease-associated Eag1 mutant proteins.ConclusionOverall these results highlight a chaperone-like role of endogenous 14-3-3 proteins in regulating Eag1 protein homeostasis, as well as a therapeutic potential of 14-3-3 modulators in correcting defective protein expression of disease-causing Eag1 mutants.

A hydrophobic nexus at the PAS-cap/globular PAS/CNPhd interface tunes deactivation kinetics of hERG channels

Slow deactivation is a critical biophysical property of human ether-à -go-go related gene (hERG) channels, mediating the cardiac current IKr during repolarization of the ventricular action potential. Disruption of interactions between the intracellular N-terminal Per-Arnt-Sim (PAS) and C-terminal cyclic nucleotide-binding homology (CNBh) domains can impair slow deactivation and is associated with long QT syndrome (LQTS). The PAS-cap (residues 1-25), an essential mediator of slow deactivation, is composed of an unstructured segment (residues 1-12) and an amphipathic α-helix (residues 13-25).

PAS and CNBH domain interactions in hERG channels investigated with surface plasmon resonance

Human ether-á-go-go-related gene (hERG) potassium channels play a key role in the regulation of cardiac repolarization, neuronal excitability, and tumorigenesis. hERG channels contain N-terminal Per-Arnt-Sim (PAS) and C-terminal cyclic nucleotide-binding homology (CNBH) domains. Structural, functional and fluorescence-based studies established that the hERG PAS and CNBH domains from adjacent subunits interact. Moreover, many long-QT syndrome (LQTS)-linked mutations are located at the interface between these domains.

Investigation of PAS and CNBH domain interactions in hERG channels and effects of long-QT syndrome-causing mutations with surface plasmon resonance

Human ether-á-go-go-related gene (hERG) channels are key regulators of cardiac repolarization, neuronal excitability, and tumorigenesis. hERG channels contain N-terminal Per-Arnt-Sim (PAS) and C-terminal cyclic nucleotide-binding homology (CNBH) domains with many long-QT syndrome (LQTS)-causing mutations located at the interface between these domains. Despite the importance of PAS/CNBH domain interactions, little is known about their affinity. Here, we used the surface plasmon resonance (SPR) technique to investigate interactions between isolated PAS and CNBH domains and the effects of LQTS-causing mutations R20G, N33T, and E58D, located at the PAS/CNBH domain interface, on these interactions. We determined that the affinity of the PAS/CNBH domain interactions was ∼1.4 μM. R20G and E58D mutations had little effect on the domain interaction affinity, while N33T abolished the domain interactions. Interestingly, mutations in the intrinsic ligand, a conserved stretch of amino acids occupying the beta-roll cavity in the CNBH domain, had little effect on the affinity of PAS/CNBH domain interactions. Additionally, we determined that the isolated PAS domains formed oligomers with an interaction affinity of ∼1.6 μM. Coexpression of the isolated PAS domains with the full-length hERG channels or addition of the purified PAS protein inhibited hERG currents. These PAS/PAS interactions can have important implications for hERG function in normal and pathological conditions associated with increased surface density of channels or interaction with other PAS-domain-containing proteins. Taken together, our study provides the first account of the binding affinities for wild-type and mutant hERG PAS and CNBH domains and highlights the potential functional significance of PAS/PAS domain interactions.

Conformation-sensitive antibody reveals an altered cytosolic PAS/CNBh assembly during hERG channel gating

The human ERG (hERG) K+ channel has a crucial function in cardiac repolarization, and mutations or channel block can give rise to long QT syndrome and catastrophic ventricular arrhythmias. The cytosolic assembly formed by the Per-Arnt-Sim (PAS) and cyclic nucleotide binding homology (CNBh) domains is the defining structural feature of hERG and related KCNH channels. However, the molecular role of these two domains in channel gating remains unclear. We have previously shown that single-chain variable fragment (scFv) antibodies can modulate hERG function by binding to the PAS domain. Here, we mapped the scFv2.12 epitope to a site overlapping with the PAS/CNBh domain interface using NMR spectroscopy and mutagenesis and show that scFv binding in vitro and in the cell is incompatible with the PAS interaction with CNBh. By generating a fluorescently labeled scFv2.12, we demonstrate that association with the full-length hERG channel is state dependent. We detect Förster resonance energy transfer (FRET) with scFv2.12 when the channel gate is open but not when it is closed. In addition, state dependence of scFv2.12 FRET signal disappears when the R56Q mutation, known to destabilize the PAS-CNBh interaction, is introduced in the channel. Altogether, these data are consistent with an extensive structural alteration of the PAS/CNBh assembly when the cytosolic gate opens, likely favoring PAS domain dissociation from the CNBh domain.

A Hydrophobic Patch in the PAS-cap Alpha-Helix Is Critical for Slow Deactivation in HERG Channels

Slow deactivation is a critical biophysical and physiological property of human ether-à-go-go-related gene (hERG) channels mediating the cardiac repolarizing current IKr. Disruption of direct interactions between the intracellular N-terminal eag domain and the C-terminal cyclic nucleotide binding homology domain (CNBhD) can impair slow repolarization and are associated with disease. The eag domain is multipartite: it comprises an N-terminal Per-Arnt-Sim (PAS) cap (residues 1-25) and globular PAS domain (residues 26-135).

Magnesium efflux from Drosophila Kenyon cells is critical for normal and diet-enhanced long-term memory.

Dietary magnesium (Mg2+) supplementation can enhance memory in young and aged rats. Memory-enhancing capacity was largely ascribed to increases in hippocampal synaptic density and elevated expression of the NR2B subunit of the NMDA-type glutamate receptor. Here we show that Mg2+ feeding also enhances long-term memory in Drosophila. Normal and Mg2+-enhanced fly memory appears independent of NMDA receptors in the mushroom body and instead requires expression of a conserved CNNM-type Mg2+-efflux transporter encoded by the unextended (uex) gene. UEX contains a putative cyclic nucleotide-binding homology domain and its mutation separates a vital role for uex from a function in memory. Moreover, UEX localization in mushroom body Kenyon cells (KCs) is altered in memory-defective flies harboring mutations in cAMP-related genes. Functional imaging suggests that UEX-dependent efflux is required for slow rhythmic maintenance of KC Mg2+. We propose that regulated neuronal Mg2+ efflux is critical for normal and Mg2+-enhanced memory.

Toward in vivo-relevant hERG safety assessment and mitigation strategies based on relationships between non-equilibrium blocker binding, three-dimensional channel-blocker interactions, dynamic occupancy, dynamic exposure, and cellular arrhythmia.

The human ether-a-go-go-related voltage-gated cardiac ion channel (commonly known as hERG) conducts the rapid outward repolarizing potassium current in cardiomyocytes (IKr). Inadvertent blockade of this channel by drug-like molecules represents a key challenge in pharmaceutical R&D due to frequent overlap between the structure-activity relationships of hERG and many primary targets. Building on our previous work, together with recent cryo-EM structures of hERG, we set about to better understand the energetic and structural basis of promiscuous blocker-hERG binding in the context of Biodynamics theory. We propose a two-step blocker binding process consisting of: The initial capture step: diffusion of a single fully solvated blocker copy into a large cavity lined by the intra-cellular cyclic nucleotide binding homology domain (CNBHD). Occupation of this cavity is a necessary but insufficient condition for ion current disruption.The IKr disruption step: translocation of the captured blocker along the channel axis, such that: The head group, consisting of a quasi-rod-shaped moiety, projects into the open pore, accompanied by partial de-solvation of the binding interface.One tail moiety packs along a kink between the S6 helix and proximal C-linker helix adjacent to the intra-cellular entrance of the pore, likewise accompanied by mutual de-solvation of the binding interface (noting that the association barrier is comprised largely of the total head + tail group de-solvation cost).Blockers containing a highly planar moiety that projects into a putative constriction zone within the closed channel become trapped upon closing, as do blockers terminating prior to this region.A single captured blocker copy may conceivably associate and dissociate to/from the pore many times before exiting the CNBHD cavity. Lastly, we highlight possible flaws in the current hERG safety index (SI), and propose an alternate in vivo-relevant strategy factoring in: Benefit/risk.The predicted arrhythmogenic fractional hERG occupancy (based on action potential (AP) simulations of the undiseased human ventricular cardiomyocyte).Alteration of the safety threshold due to underlying disease.Risk of exposure escalation toward the predicted arrhythmic limit due to patient-to-patient pharmacokinetic (PK) variability, drug-drug interactions, overdose, and use for off-label indications in which the hERG safety parameters may differ from their on-label counterparts.

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-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.

Mg2+-ATP Sensing in CNNM, a Putative Magnesium Transporter

The family of cystathionine-β-synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) is composed of four integral membrane proteins associated with Mg2+ transport. Structurally, CNNMs contain large cytosolic regions composed of a CBS-pair and a cyclic nucleotide-binding homology (CNBH) domain. How these regulate Mg2+ transport activity is unknown. Here, we determined the crystal structures of cytosolic fragments in two conformations: Mg2+-ATP-analog bound and ligand free. The structures reveal open and closed conformations with functionally important contacts not observed in structures of the individual domains. We also identified a second Mg2+-binding region in the CBS-pair domain and a different dimerization interface for the CNBH domain. Analytical ultracentrifugation and isothermal titration calorimetry experiments revealed a tight correlation between Mg2+-ATP binding and protein dimerization. Mutations that blocked either function prevented cellular Mg2+ efflux activity. The results suggest Mg2+ efflux is regulated by conformational changes associated with Mg2+-ATP binding to CNNM CBS-pair domains.

Identification of undecylenic acid as EAG channel inhibitor using surface plasmon resonance-based screen of KCNH channels

BackgroundKCNH family of potassium channels is responsible for diverse physiological functions ranging from the regulation of neuronal excitability and cardiac contraction to the regulation of cancer progression. KCNH channels contain a Per-Arn-Sim (PAS) domain in their N-terminal and cyclic nucleotide-binding homology (CNBH) domain in their C-terminal regions. These intracellular domains shape the function of KCNH channels and are important targets for drug development.MethodsHere we describe a surface plasmon resonance (SPR)-based screening method aimed in identifying small molecule binders of PAS and CNBH domains for three KCNH channel subfamilies: ether-à-go-go (EAG), EAG-related gene (ERG), and EAG-like K+ (ELK). The method involves purification of the PAS and CNBH domains, immobilization of the purified domains on the SPR senor chip and screening small molecules in a chemical library for binding to the immobilized domains using changes in the SPR response as a reporter of the binding. The advantages of this method include low quantity of purified PAS and CNBH domains necessary for the implementation of the screen, direct assessment of the small molecule binding to the PAS and CNBH domains and easiness of assessing KCNH subfamily specificity of the small molecule binders.ResultsUsing the SPR-based method we screened the Spectrum Collection Library of 2560 compounds against the PAS and CNBH domains of the three KCNH channel subfamilies and identified a pool of small molecules that bind to the PAS or CNBH domains. To further evaluate the effectiveness of the screen we tested the functional effect of one of the identified mEAG PAS domain specific small molecule binders on currents recorded from EAG channels. Undecylenic acid inhibited currents recorded from EAG channels in a concentration-dependent manner with IC50 of ~ 1 μM.ConclusionOur results show that the SPR-based method is well suited for identifying small molecule binders of KCNH channels and can facilitate drug discovery for other ion channels as well.

Identifying Novel KCNH Channel Ligands with Surface Plasmon Resonance Method

KCNH family of potassium channels is responsible for diverse physiological functions ranging from the regulation of neuronal excitability and cardiac contraction to the regulation of cancer progression. KCNH channels contain a Per-Arn-Sim (PAS) domain in their N-terminal and cyclic nucleotide-binding homology (CNBH) domain in their C-terminal regions. These intracellular domains shape the function and contribute to the unique properties of KCNH channels. The structures of PAS and CNBH domains suggest that they harbor potential binding sites for small molecules. Here we describe a surface plasmon resonance (SPR)-based high-throughput screening method aimed in identifying small molecule binders for PAS and CNBH domains of KCNH channels. The method involves purification of PAS and CNBH domains of KCNH channels, immobilization of the purified domains on the SPR senor chip and screening small molecules in a chemical library for binding to the immobilized domains using changes in the SPR-angle as a reporter of the binding. The advantages of this method include low quantity of purified PAS and CNBH domains necessary for the implementation of the screen, direct assessment of the binding between the PAS and CNBH domains and easiness of assessing KCNH subfamily specificity of the small molecule binders. To demonstrate the effectiveness of the method we screened small molecules in the Spectrum Collection Library and identified a small molecule binder that binds to the PAS domains in a subfamily specific manner. These results show that the SPR-based method is well suited for identifying small molecule binders of KCNH channels.

Conformational Dynamics of the Intrinsic Ligand in the CNBHD of the Voltage-Gated Potassium Channel hERG

Human ether-á-go-go related gene (hERG) voltage-activated potassium channels are critical for cardiac excitability. Characteristic slow closing (deactivation) in hERG is regulated by a direct interaction between the N-terminal Per-Arnt-Sim (PAS) domain and the C-terminal cyclic nucleotide binding homology domain (CNBHD). An intrinsic ligand in hERG is located at the PAS-CNBHD interface, but its mechanism is not well understood. Our recent work with co-expressed channel fragments in which the hERG PAS domain fused to CFP was expressed in trans with the ‘core’ of the hERG channel (i.e. a channel lacking the PAS domain that is fused to Citrine at the C-terminus, hERG ΔPAS-Citrine) showed that deactivation gating was disrupted (was made faster) by intrinsic ligand mutations and that the activation time course of the channel was perturbed (was made faster), indicating that the intrinsic ligand is necessary for the functional and structural interaction of the PAS domain and the CNBHD. To better understand the dynamics for intrinsic ligand regulation of hERG gating, we incorporated the fluorescent non-canonical amino acid ANAP using amber codon suppression at the intrinsic ligand, and engineered dihistidines that were structurally adjacent to this ANAP, to allow us to probe a change (1) using an environmentally-dependent spectral shift of ANAP and/or (2) using transition metal FRET. This technique will determine how the intrinsic ligand at the PAS-CNBHD interface may undergo conformational changes with voltage to regulate channel deactivation gating.

Dynamics of the PAS Domain and Cyclic Nucleotide-Binding Homology Domain Interaction Probed with a Fluorescent Noncanonical Amino Acid (L-ANAP) in HERG Potassium Channels

Dynamics of the PAS Domain and Cyclic Nucleotide-Binding Homology Domain Interaction Probed with a Fluorescent Noncanonical Amino Acid (L-ANAP) in HERG Potassium Channels

The cyclic nucleotide–binding homology domain of the integral membrane protein CNNM mediates dimerization and is required for Mg2+ efflux activity

Proteins of the cyclin M family (CNNMs; also called ancient conserved domain proteins, or ACDPs) are represented by four integral membrane proteins that have been proposed to function as Mg2+ transporters. CNNMs are associated with a number of genetic diseases affecting ion movement and cancer via their association with highly oncogenic phosphatases of regenerating liver (PRLs). Structurally, CNNMs contain an N-terminal extracellular domain, a transmembrane domain (DUF21), and a large cytosolic region containing a cystathionine-β-synthase (CBS) domain and a putative cyclic nucleotide-binding homology (CNBH) domain. Although the CBS domain has been extensively characterized, little is known about the CNBH domain. Here, we determined the first crystal structures of the CNBH domains of CNNM2 and CNNM3 at 2.6 and 1.9 Å resolutions. Contrary to expectation, these domains did not bind cyclic nucleotides, but mediated dimerization both in crystals and in solution. Analytical ultracentrifugation experiments revealed an inverse correlation between the propensity of the CNBH domains to dimerize and the ability of CNNMs to mediate Mg2+ efflux. CNBH domains from active family members were observed as both dimers and monomers, whereas the inactive member, CNNM3, was observed only as a dimer. Mutational analysis revealed that the CNBH domain was required for Mg2+ efflux activity of CNNM4. This work provides a structural basis for understanding the function of CNNM proteins in Mg2+ transport and associated diseases.