Coiled-coil Helix Research Articles

IP6 Does Not Induce Binding between Coiled-Coil Helices or between the N-Term ARDS and Coiled-Coil

Transient Receptor Potential Ankyrin type 1 (TRPA1) is a cation-selective ion channel that is activated by several noxious compounds and by reduced temperature. Given its role in noxious sensation and inflammation, TRPA1 has become an active drug target for the treatment of pain and inflammation-related ailments such as itch and cough. Although the cryo-EM structure of TRPA1 has provided a starting point in understanding channel activation, the structural mechanisms remain elusive. In particular, an N-terminal Ankyrin Repeat Domain (ARD) contains a highly reactive cysteine that activates TRPA1 demonstrating the importance of this region in channel function. Furthermore, it was hypothesized that a tetrameric C-terminal coiled-coil was stabilized by a polyphosphate compound, IP6, which would account for the requirement of polyphosphates in excised patch electrophysiology experiments recording TRPA1 activity. Here, we used biochemical and biophysical approaches of isolated cytoplasmic domains from human TRPA1 to probe the role of IP6 on the oligomerization of N-terminal ARD and the C-terminal coiled-coil. Surprisingly, we found that oligomerization of the C-terminal coiled-coil was indistinguishable in the absence and presence of IP6. IP6 similarly had no discernable effect on binding of the ARD to the C-terminal coiled-coil. Our data suggest that the role of IP6 in TRPA1 activation likely involves mechanisms not yet considered.

CHTM1, a novel metabolic marker deregulated in human malignancies.

A better understanding of the link between cellular metabolism and tumorigenesis is needed. Here, we report characterization of a novel protein named Coiled-coil Helix Tumor and Metabolism 1 (CHTM1). We have found that CHTM1 is associated with cancer and cellular metabolism. CHTM1 localizes to mitochondria and cytosol, and its deficiency in cancer cells results in decreased mitochondrial oxygen consumption and ATP levels as well as oxidative stress indicating mitochondrial dysfunction. CHTM1-deficient cancer cells display poor growth under glucose/glutamine-deprived conditions, whereas cells expressing increased levels of exogenous CHTM1 exhibit enhanced proliferation and survival under similar conditions. CHTM1 deficiency also leads to defects in lipid metabolism resulting in fatty acid accumulation, which explains poor growth of CHTM1-deficient cells under glucose/glutamine deprivation since nutrient deprivation increases dependency on lipids for energy generation. We also demonstrate that CHTM1 mediates its effect via the PKC, CREB and PGC-1alpha signaling axis, and cytosolic accumulation of CHTM1during nutrient deprivation appears to be important for its effect on cellular signaling events. Furthermore, analyses of tissue specimens from 71 breast and 97 colon cancer patients show CHTM1 expression to be upregulated in the majority of tumor specimens representing these malignancies. Collectively, our findings are highly significant because CHTM1 is a novel metabolic marker that is important for the growth of tumorigenic cells under limiting nutrient supplies and thus, links cellular metabolism and tumorigenesis.

Abstract 4402: CHTM1, a novel protein linked to cellular metabolism and human malignancy

Abstract Several lines of emerging evidence indicate that alterations in metabolism are linked to pathogenesis of human malignancies. However, more studies are needed to completely understand the link between tumorigenesis and metabolism. In this context, we have characterized a novel protein named CHTM1 (Coiled-coil Helix Tumor and Metabolism 1) that appears to be associated with cancer and cellular metabolism. Per our findings, CHTM1 was present in cytosol as well as mitochondria. CHTM1 knockdown in human cancer cell lines caused oxidative stress, and decrease in cellular oxygen consumption and mitochondrial ATP levels, suggesting mitochondrial dysfunction. In view of the importance of mitochondria to nutrient stress, the effect of CHTM1 deficiency on human cancer cells in glucose-deprived conditions was also analyzed. The results indicated that CHTM1-deficient cells became more sensitive to glucose deprivation and exhibited altered cellular metabolism involving down regulation of PKC-CREB-PGC1 alpha signaling events. CHTM1 expression was also found to be increased in patient samples representing breast, colon and lung cancers when compared to their respective matching normal tissues. Based on our findings, we propose CHTM1 to be a valuable tumor marker that can also be developed as a target for novel therapeutics particularly those that exploit cellular metabolism to mediate their effects. Citation Format: Mansi Babbar, Ying Huang, M Saeed Sheikh. CHTM1, a novel protein linked to cellular metabolism and human malignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4402. doi:10.1158/1538-7445.AM2017-4402

Oligomeric and Thermal Stability of TRPA1 Coiled-Coil Domain by Polyphosphates

Human Transient Receptor Potential Ankyrin 1 (TRPA1) is a cation channel involved in the sensation of pain that is regulated by multiple stimuli including temperature, a wide range of irritants, and oxidative stimuli. The importance of TRPA1 is underscored by the numerous studies of the channel as a target for novel pain therapies. The recent cyro-EM structure (Paulsen et al, 2015) provides a strong framework in which to understand the mechanisms underlying functional activation. The structure of TRPA1 revealed the presence of a C-terminal coiled-coil domain that was hypothesized to require polyphosphates for oligomeric structural stability. This is consistent with the requirement of polyphosphates for functional channel expression assessed via electrophysiology (Kim and Cavanaugh, 2007). Furthermore, it has been speculated that the unfolding of the TRPA1 coiled-coil helices in response to temperature could be involved in thermal gating, as has been observed in bacterial sodium channels (Arrigoni et al, 2016). Here we examine the biochemical and biophysical properties of the coiled-coil of TRPA1 to determine the role of polyphosphates in oligomerization and their contributions to thermal stability.

Crystal Structure of Human General Transcription Factor TFIIE at Atomic Resolution

In eukaryotes, RNA polymerase II requires general transcription factors to initiate mRNA transcription. TFIIE subunits α and β form a heterodimer and recruit TFIIH to complete the assembly of the pre-initiation complex. Here, we have determined the crystal structure of human TFIIE at atomic resolution. The N-terminal half of TFIIEα forms an extended winged helix (WH) domain with an additional helix, followed by a zinc-finger domain. TFIIEβ contains the WH2 domain, followed by two coiled-coil helices intertwining with TFIIEα. We also showed that TFIIEα binds to TFIIEβ with nanomolar affinity using isothermal titration calorimetry. In addition, mutations on the residues involved in the interactions resulted in severe growth defects in yeast. Lack of the C-terminal region of yeast TFIIEβ causes a mild growth defect in vivo. These findings provide a structural basis for understanding the functional mechanisms of TFIIE in the context of pre-initiation complex formation and transcription initiation.

A De Novo Virus-Like Topology for Synthetic Virions.

A de novo topology of virus-like assembly is reported. The design is a trifaceted coiled-coil peptide helix, which self-assembles into ultrasmall, monodisperse, anionic virus-like shells that encapsulate and transfer both RNA and DNA into human cells. Unlike existing artificial systems, these shells share the same physical characteristics of viruses being anionic, nonaggregating, abundant, hollow, and uniform in size, while effectively mediating gene silencing and transgene expression. These are the smallest virus-like structures reported to date, both synthetic and native, with the ability to adapt and transfer small and large nucleic acids. The design thus offers a promising solution for engineering bespoke artificial viruses with desired functions.

The N-terminal domain plays a crucial role in the structure of a full-length human mitochondrial Lon protease.

Lon is an essential, multitasking AAA+ protease regulating many cellular processes in species across all kingdoms of life. Altered expression levels of the human mitochondrial Lon protease (hLon) are linked to serious diseases including myopathies, paraplegia, and cancer. Here, we present the first 3D structure of full-length hLon using cryo-electron microscopy. hLon has a unique three-dimensional structure, in which the proteolytic and ATP-binding domains (AP-domain) form a hexameric chamber, while the N-terminal domain is arranged as a trimer of dimers. These two domains are linked by a narrow trimeric channel composed likely of coiled-coil helices. In the presence of AMP-PNP, the AP-domain has a closed-ring conformation and its N-terminal entry gate appears closed, but in ADP binding, it switches to a lock-washer conformation and its N-terminal gate opens, which is accompanied by a rearrangement of the N-terminal domain. We have also found that both the enzymatic activities and the 3D structure of a hLon mutant lacking the first 156 amino acids are severely disturbed, showing that hLon’s N-terminal domains are crucial for the overall structure of the hLon, maintaining a conformation allowing its proper functioning.

A Novel Site of Competitive PIP2 and Calmodulin Interaction to KCNQ1 C-Terminus Helix B is Crucial for IKs Channel Activity

KCNQ1 and KCNE1 co-assembly generates the IKS potassium current, which is crucial to the cardiac action potential repolarization. Mutations in their corresponding genes cause cardiac arrhythmias. In the KCNQ1 C-terminus (CT), proximal helices A and B form sites for calmodulin (CaM) binding, whereas distal coiled-coil helices C and D are necessary for subunit tetramerization. Studies identified basic residues in KCNQ1 at S2-S3 and S4-S5 intracellular linkers and proximal CT as PIP2 binding sites. Our recent crystallographic data showed that CaM embraces the two proximal anti-parallel helices B and A with its calcified N-lobe and apo C-lobe, respectively. Here we identified a novel site of competitive PIP2 and calmodulin interaction to helix B that is crucial for IKS channel activity. PIP2 competes with CaM binding to purified His-tagged KCNQ1 CT in the presence of Ca2+ only (IC50 = 39 µM). Conversely, recombinant WT Ca2+-CaM or CaM3,4 (IC50 = 1 µM) but neither WT apoCaM nor CaM1,2 compete with PIP2 binding to purified His-tagged KCNQ1 CT. Recombinant WT Ca2+-CaM or CaM3,4 but not WT apoCaM or CaM1,2 in the patch pipet significantly attenuate the decrease in IKS current resulting from PIP2 breakdown by Dr-VSP. Molecular docking, dynamic simulation and subsequent biochemical and electrophysiological validation experiments indicate that K526 and K527 in helix B form a novel and crucial site for competitive PIP2 and calmodulin interaction. Our data suggest that upon PIP2 breakdown (e.g. following GPCR-Gq signaling), PIP2 unbinds from helix B and allows the calcified CaM N-lobe to replace it, which guaranties the maintenance of the channel open state in front of stressful PIP2 depletion events.

Construction of a tissue-specific transcription factor-tethered extracellular matrix protein via coiled-coil helix formation.

Tissue-specific transcription factors are key regulators of cellular differentiation. Previously, we succeeded in introducing basic helix-loop-helix tissue-specific transcription factor proteins into cells to induce cellular differentiation. Based on these results, we decided to focus on the use of tissue-specific transcription factor proteins in the construction of biomaterials. In this proof-of-concept study, we demonstrate the construction of a tissue-specific transcription factor-tethered extracellular matrix protein. Here, the tissue-specific transcription factor Olig2 was tethered to a designed artificial extracellular matrix protein via coiled-coil helix formation. Tethered Olig2 was introduced into mouse embryonic carcinoma P19 cells attached to our designed extracellular matrix protein, and was shown to exhibit the ability to induce neural differentiation.

Human mitochondrial MIA40 (CHCHD4) is a component of the Fe-S cluster export machinery.

Mitochondria play an essential role in synthesis and export of iron-sulfur (Fe-S) clusters to other sections of a cell. Although the mechanism of Fe-S cluster synthesis is well elucidated, information on the identity of the proteins involved in the export pathway is limited. The present study identifies hMIA40 (human mitochondrial intermembrane space import and assembly protein 40), also known as CHCHD4 (coiled-coil-helix-coiled-coil-helix domain-containing 4), as a component of the mitochondrial Fe-S cluster export machinery. hMIA40 is an iron-binding protein with the ability to bind iron invivo and invitro. hMIA40 harbours CPC (Cys-Pro-Cys) motif-dependent Fe-S clusters that are sensitive to oxidation. Depletion of hMIA40 results in accumulation of iron in mitochondria concomitant with decreases in the activity and stability of Fe-S-containing cytosolic enzymes. Intriguingly, overexpression of either the mitochondrial export component or cytosolic the Fe-S cluster assembly component does not have any effect on the phenotype of hMIA40-depleted cells. Taken together, our results demonstrate an indispensable role for hMIA40 for the export of Fe-S clusters from mitochondria.

Highlights of This Issue

CHCHD2 encodes a coiled-coil helix coiled-coil helix domain-containing protein that maps close to EGFR, and it is recurrently coamplified in non–small cell lung cancer (NSCLC), which accounts for more deaths per year than any other cancer. CHCHD2 protein is consistently upregulated in NSCLC, and its knockdown impairs cell proliferation, migration, and mitochondrial respiration. CHCHD2 is predominantly mitochondrial, and it is required for the stable assembly of oxidative phosphorylation components. The CHCHD2 interactome also includes an oncogenic transcription factor. Hence, CHCHD2 appears to function through mitochondrial and nuclear interactions, and, along with EGFR, appears to be a driver of 7p11.2 amplification and the cancer phenotype.Anticancer drugs targeting microtubule dynamics are widely prescribed for a broad range of cancers. However, drug resistance and severe side effects in treated patients limit their effectiveness. Lindström and colleagues show that citral dimethyl acetal (CDA), a citral analogue that specifically targets the nuclear activity of γ-tubulin, is a member of the tubulin family but has no effect on microtubule dynamics. CDA kills tumor cells with an impaired retinoblastoma (RB1) tumor suppressor signaling pathway without affecting healthy cells. CDA's in vivo antitumorigenic activity paves the way for the development of a novel broad-range, targeted anticancer therapy that causes fewer side effects.Transglutaminase-2 (TG2) is a vital cross-linking enzyme in the tumor microenvironment that has been linked to cancer progression, but its precise role appears to be context dependent. Cellura and colleagues found that TG2 inhibits invasion of colorectal cancer cells in vitro. Using a cell model of metastasis and analysis in sections taken from patients, a novel mechanism is described by which TG2 is downregulated in advanced colorectal cancer. Amplification of chromosome 13 leads to enhanced levels of miR-19 in metastatic cells, inhibiting TG2 to promote invasion. This study provides an important link between chromosomal instability, miRNA expression, and TG2 status, with functional effects on colorectal cancer cell behavior.The hypermethylated in cancer 1 (HIC1) gene encodes a sequence-specific transcriptional repressor that is frequently silenced in many solid tumors. Janeckova and colleagues used a conditional knock-out of Hic1 to identify genes regulated by this repressor. Hic1 ablation in primary fibroblasts and intestinal epithelia resulted in upregulation of Toll-like receptor 2 (TLR2), a critical mediator of prosurvival signal transduction pathways. In chemically induced colon cancer, Hic1 loss caused formation of highly proliferative Tlr2-positive neoplastic lesions. This study reveals that the tumor-suppressive function of Hic1 is related to its inhibitory action on signaling mediated by the Tlr2 receptor present on tumor cells.

MNRR1 (formerly CHCHD2) is a bi-organellar regulator of mitochondrial metabolism

Our understanding of stress-associated regulatory mechanisms for mitochondria remains incomplete. We now report a new regulator of mitochondrial metabolism, the coiled-coil–helix–coiled-coil–helix domain-containing protein 2 (CHCHD2) which, based on the functionality described here, is renamed MNRR1 (Mitochondria Nuclear Retrograde Regulator 1). It functions in a novel way by acting in two cellular compartments, mitochondria and nucleus. In normally growing cells most MNRR1 is located in mitochondria; during stress most MNRR1 is now located in the nucleus. MNRR1 is imported to the mitochondrial intermembrane space by a Mia40-mediated pathway, where it binds to cytochrome c oxidase (COX). This association is required for full COX activity. Decreased MNRR1 levels produce widespread dysfunction including reduced COX activity, membrane potential, and growth rate, and increased reactive oxygen species and mitochondrial fragmentation. In the nucleus, MNRR1 acts as a transcription factor, one of whose targets is the COX subunit 4 isoform, COX4I2, which is transcriptionally stimulated by hypoxia. This MNRR1-mediated stress response may provide an important survival mechanism for cells under conditions of oxidative or hypoxic stress, both in the acute phase by altering mitochondrial oxygen utilization and in the chronic phase by promoting COX remodeling.

A mitochondrial origin for frontotemporal dementia and amyotrophic lateral sclerosis through CHCHD10 involvement.

Mitochondrial DNA instability disorders are responsible for a large clinical spectrum, among which amyotrophic lateral sclerosis-like symptoms and frontotemporal dementia are extremely rare. We report a large family with a late-onset phenotype including motor neuron disease, cognitive decline resembling frontotemporal dementia, cerebellar ataxia and myopathy. In all patients, muscle biopsy showed ragged-red and cytochrome c oxidase-negative fibres with combined respiratory chain deficiency and abnormal assembly of complex V. The multiple mitochondrial DNA deletions found in skeletal muscle revealed a mitochondrial DNA instability disorder. Patient fibroblasts present with respiratory chain deficiency, mitochondrial ultrastructural alterations and fragmentation of the mitochondrial network. Interestingly, expression of matrix-targeted photoactivatable GFP showed that mitochondrial fusion was not inhibited in patient fibroblasts. Using whole-exome sequencing we identified a missense mutation (c.176C>T; p.Ser59Leu) in the CHCHD10 gene that encodes a coiled-coil helix coiled-coil helix protein, whose function is unknown. We show that CHCHD10 is a mitochondrial protein located in the intermembrane space and enriched at cristae junctions. Overexpression of a CHCHD10 mutant allele in HeLa cells led to fragmentation of the mitochondrial network and ultrastructural major abnormalities including loss, disorganization and dilatation of cristae. The observation of a frontotemporal dementia-amyotrophic lateral sclerosis phenotype in a mitochondrial disease led us to analyse CHCHD10 in a cohort of 21 families with pathologically proven frontotemporal dementia-amyotrophic lateral sclerosis. We identified the same missense p.Ser59Leu mutation in one of these families. This work opens a novel field to explore the pathogenesis of the frontotemporal dementia-amyotrophic lateral sclerosis clinical spectrum by showing that mitochondrial disease may be at the origin of some of these phenotypes.

The Mitochondrial Intermembrane Space Oxireductase Mia40 Funnels the Oxidative Folding Pathway of the Cytochrome c Oxidase Assembly Protein Cox19

Mia40-catalyzed disulfide formation drives the import of many proteins into the mitochondria. Here we characterize the oxidative folding of Cox19, a twin CX9C Mia40 substrate. Cox19 oxidation is extremely slow, explaining the persistence of import-competent reduced species in the cytosol. Mia40 accelerates Cox19 folding through the specific recognition of the third Cys in the second helical CX9C motif and the subsequent oxidation of the inner disulfide bond. This renders a native-like intermediate that oxidizes in a slow uncatalyzed reaction into native Cox19. The same intermediate dominates the pathway in the absence of Mia40, and chemical induction of an α-helical structure by trifluoroethanol suffices to accelerate productive folding and mimic the Mia40 folding template mechanism. The Mia40 role is to funnel a rough folding landscape, skipping the accumulation of kinetic traps, providing a rationale for the promiscuity of Mia40.

The spectrin family of proteins: A unique coiled-coil fold for various molecular surface properties

The spectrin superfamily is composed of proteins involved in cytolinker functions. Their main structural feature is a large central subdomain with numerous repeats folded in triple helical coiled-coils. Their similarity of sequence was considered to be low without detailed quantification of the intra- and intermolecular levels. Among the superfamily, we considered as essential to propose an overview of the surface properties of all the repeats of the five proteins of the spectrin family, namely α- and β-spectrins, α-actinin, dystrophin and utrophin. Therefore, the aim of this work was to obtain a quantitative comparison of all the repeats at both the primary sequence and the three-dimensional levels. For that purpose, we applied homology modelling methods to obtain structural models for successive and overlapping tandem repeats of the human erythrocyte α- and β-spectrins and utrophin, as previously undertaken for dystrophin, and we used the known structure of α-actinin. The matrix calculation of the pairwise similarities of all the repeat sequences and the electrostatic and hydrophobic surface properties throughout the protein family support the view that spectrins and α-actinin on one hand and utrophin and dystrophin on the other hand share some structural similarities, but a detailed molecular characterisation highlights substantial differences. The repeats within the family are far from identical, which is consistent with their multiple interactions with different cellular partners, including proteins and membrane lipids.

Coiled-Coil Helix Rotation Selects Repressing or Activating State of Transcriptional Regulator DhaR

Escherichia coli dihydroxyacetone (Dha) kinase consists of two subunits, DhaK and DhaL. Transcription of dha operon is regulated by the DhaR transcription factor and its action is under control of the kinase subunits. DhaR is activated by interaction with DhaL while it is repressed by DhaK. We have determined the structures of DhaK and DhaL bound to the tandem GAF-like and PAS domains of the DhaR, providing an architectural model for how GAF/PAS tandem domains work together in binding protein partners. The structures reveal a mechanism of opposite transcriptional regulation by the DhaK and DhaL subunits. The kinase subunits interface with DhaR through surfaces that partially overlap with their active sites, allowing sensing of ATP- versus ADP-loaded DhaL subunit and also precluding a ternary complex between DhaK-DhaL and DhaR. The rotation of helices within the DhaR coiled-coil linker upon DhaL binding provides the mechanism for transmitting the binding signal from the GAF/PAS domains to the C-terminal DNA-binding domain.

Structural Basis for the Assembly of Kinesin-5 into Bipolar Anti-Parallel Tetramers

Cell division is driven by organized mitotic spindles and requires diverse groups of molecular microtubule-based kinesin motor proteins. Kinesin-5 motors form a unique and conserved class of tetrameric bipolar motor proteins, consisting of dual motor domain sets oriented at opposite ends of a central rod-like structure. The bipolar organization is critical for kinesin-5 to crosslink and slide adjacent anti-parallel microtubules in opposite directions required for spindle elongation in anaphase. Here we present the atomic structure of Drosphila kinesin-5, KLF61F, bipolar assembly (BASS) domain, which explains how four molecules assemble to form a bipolar functional motor. Instead of the predicted dual traversing parallel coiled-coils, the BASS structure reveals a novel 26nm long antiparallel 4-helical bundle filament, where the basic assembly unit is an anti-parallel coiled-coil that originate from opposite ends of the bipolar structure, and folds onto a second unit in an anti-parallel manner. A striated pattern of hydrophobic and hydrophilic pockets precisely oriented monomers within the BASS tetramer to form this bipolar arrangement. Strikingly, the BASS N-termini undergo a helical exchange from anti-parallel bundle in the center to form homo-dimeric parallel helical coiled-coils at the poles. These parallel BASS N-termini are 100 degrees rotated compared to N-termini emerging from the opposite pole, providing a physical explanation for the known kinesin-5 preference for sliding anti-parallel versus parallel microtubules. We have generated a structural model for the kinesin-5 tetramer using the BASS structures and all electron microscopy that explains key features of the kinesin-5-driven sliding filament mechanism.

Backbone and side-chain 1H, 15N and 13C resonance assignments of the microtubule-binding domain of yeast cytoplasmic dynein in the high and low-affinity states

Cytoplasmic dynein is a motor protein that walks toward the minus end of microtubules (MTs) by utilizing the energy of ATP hydrolysis. The heavy chain of cytoplasmic dynein contains the microtubule-binding domain (MTBD). Switching of MTBD between high and low affinity states for MTs is crucial for processive movement of cytoplasmic dynein. Previous biochemical studies demonstrated that the affinity of MTBD is regulated by the AAA+ family ATPase domain, which is separated by 15 nm long coiled-coil helix. In order to elucidate the structural basis of the affinity switching mechanism of MTBD, we designed two MTBD constructs, termed MTBD-High and MTBD-Low, which are locked in high and low affinity state for MTs, respectively, by introducing a disulfide bond between the coiled-coil helix. Here, we established the backbone and side-chain assignments of MTBD-High and MTBD-Low for further structural analyses.

Structures of Drosophila Cryptochrome and Mouse Cryptochrome1 Provide Insight into Circadian Function

Drosophila cryptochrome (dCRY) is a FAD-dependent circadian photoreceptor, whereas mammalian cryptochromes (CRY1/2) are integral clock components that repress mCLOCK/mBMAL1-dependent transcription. We report crystal structures of full-length dCRY, a dCRY loop deletion construct, and the photolyase homology region of mouse CRY1 (mCRY1). Our dCRY structures depict Phe534 of the regulatory tail in the same location as the photolesion in DNA-repairing photolyases and reveal that the sulfur loop and tail residue Cys523 plays key roles in the dCRY photoreaction. Our mCRY1 structure visualizes previously characterized mutations, an NLS, and MAPK and AMPK phosphorylation sites. We show that the FAD and antenna chromophore-binding regions, a predicted coiled-coil helix, the C-terminal lid, and charged surfaces are involved in FAD-independent mPER2 and FBXL3 binding and mCLOCK/mBMAL1 transcriptional repression. The structure of a mammalian cryptochrome1 protein may catalyze the development of CRY chemical probes and the design of therapeutic metabolic modulators.

The Distal Kcne1 C-Terminus is Crucial for Yotiao Mediated Pka-Dependent Phosphorylation of KCNQ1

The assembly of KCNQ1 with KCNE1 produces the IKSpotassium current that is crucial for the late repolarization of the cardiac action potential. Mutations in either KCNQ1 or KCNE1 genes produce the long QT or short QT syndromes, which are genetically heterogeneous cardiovascular diseases, characterized by ventricular or atrial arrhythmias. The scaffolding A-kinase anchoring protein (AKAP) Yotiao brings together PKA, PP1, PDE4D3, AC9, and the IKS channel complex to achieve regulation following beta adrenergic stimulation.We recently showed that the distal KCNE1 C-terminus interacts with the coiled-coil helix C of KCNQ1 C-terminus. Here we examined the effect of LQT mutations located at this C-terminal interface of the two subunits. Four KCNQ1 LQT mutations located at helix C, S546L, K557E, R555C, and R562M impaired the interaction with KCNE1 C-terminus and produced a drastic reduction in IKS current density mostly caused by a right-shift of the voltage-dependence of channel activation. A much weaker PIP2 binding paralleled the decrease in IKS current density. The KCNE1 LQT mutation, P127T, situated at the distal C-terminus weakened the interaction with KCNQ1 helix C and caused a 40% decrease in IKS current density but with no shift of the voltage-dependence of channel activation. Interestingly, the KCNE1 mutant P127T markedly reduced the cAMP-dependent Yotiao-mediated upregulation of IKS current.While the P127T mutation did not alter the ability of KCNQ1 to interact with Yotiao, it strongly disrupted KCNQ1 phosphorylation of S27, in the presence of Yotiao and cAMP. Similar results were found with a deletion of the distal KCNE1 C-terminus.(del 109-129). These results suggest that the distal KCNE1 C-terminus, probably via its interaction with the coiled-coil helix C, is a crucial determinant for the functional modulation of KCNQ1 by Yotiao-mediated PKA phosphorylation.