Interaction Of Calmodulin Research Articles

Calmodulin Interaction with Gap Junction Intracellular Loop Peptides

Calmodulin Interaction with Gap Junction Intracellular Loop Peptides

Calmodulin Mutations Associated with Congenital Cardiac Disease Display Novel Biophysical and Biochemical Characteristics

Calmodulin (CaM) is a cytoplasmic multifunctional calcium (Ca2+)-binding messenger that interacts with the cardiac ryanodine receptor (RyR2), a large transmembrane Ca2+ channel that mediates Ca2+ release from the sarcoplasmic reticulum (SR) to activate cardiac muscle contraction. Recent genetic studies have reported CaM missense mutations in patients with a history of severe cardiac arrhythmogenic disorders. Herein, we have investigated the effect of four novel missense CaM mutations, identified in two patients presenting with long QT syndrome (LQTS) (N98I, D134H), and two patients with clinical features of both LQTS and catecholaminergic polymorphic ventricular tachycardia (CPVT), (D132E and Q136P), relative to the biophysical and biochemical properties of wild type CaM (CaMWT). We used CD spectroscopy to examine the thermal stability of CaMWT and mutant proteins. In the absence of Ca2+, thermodynamic values for all proteins were similar. In contrast, in the presence of Ca2+, there was a significant decrease in the stability of the five proteins following the order CaMWT> CaMN98I > CaMD132E > CaMQ136P > CaMD134H. Further Ca2+-binding studies revealed that all CaM mutations significantly reduce the Ca2+-binding affinity of CaMWT. CaMQ136P protein exhibited a ∼7-fold reduced Ca2+-binding affinity compared to CaMWT, while CaMD132E had a ∼14-fold reduction. Furthermore, biochemical analysis revealed that all four CaM mutants displayed dramatically reduced RyR2 interaction and defective modulation of [3H]ryanodine binding to RyR2, regardless of LQTS or CPVT association. Our findings confirm our previous observations suggesting that the clinical presentation of LQTS or CPVT associated with these four CaM mutations may involve both altered intrinsic Ca2+-binding as well as dysregulation of RyR2-mediated Ca2+ release via aberrant interaction of CaM with RyR2.

Direct visualization of interaction between calmodulin and connexin45.

Calmodulin (CaM) is an intracellular Ca2+ transducer involved in numerous activities in a broad Ca2+ signaling network. Previous studies have suggested that the Ca2+/CaM complex may participate in gap junction regulation via interaction with putative CaM-binding motifs in connexins; however, evidence of direct interactions between CaM and connexins has remained elusive to date due to challenges related to the study of membrane proteins. Here, we report the first direct interaction of CaM with Cx45 (connexin45) of γ-family in living cells under physiological conditions by monitoring bioluminescence resonance energy transfer. The interaction between CaM and Cx45 in cells is strongly dependent on intracellular Ca2+ concentration and can be blocked by the CaM inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7). We further reveal a CaM-binding site at the cytosolic loop (residues 164-186) of Cx45 using a peptide model. The strong binding (Kd ∼ 5 nM) observed between CaM and Cx45 peptide, monitored by fluorescence-labeled CaM, is found to be Ca2+-dependent. Furthermore, high-resolution nuclear magnetic resonance spectroscopy reveals that CaM and Cx45 peptide binding leads to global chemical shift changes of 15N-labeled CaM, but does not alter the size of the structure. Observations involving both N- and C-domains of CaM to interact with the Cx45 peptide differ from the embraced interaction with Cx50 from another connexin family. Such interaction further increases Ca2+ sensitivity of CaM, especially at the N-terminal domain. Results of the present study suggest that both helicity and the interaction mode of the cytosolic loop are likely to contribute to CaM's modulation of connexins.

Ca2+-Calmodulin and PIP2 interactions at the proximal C-terminus of Kv7 channels

ABSTRACTIn the heart, co-assembly of Kv7.1 with KCNE1 produces the slow IKS potassium current, which repolarizes the cardiac action potential and mutations in human Kv7.1 and KCNE1 genes cause cardiac arrhythmias. The proximal Kv7.1 C-terminus binds calmodulin (CaM) and phosphatidylinositol-4,5-bisphosphate (PIP2) and recently we revealed the competition of PIP2 with the calcified CaM N-lobe to a previously unidentified site in Kv7.1 helix B, also known to harbor a LQT mutation. Data indicated that PIP2 and Ca2+-CaM perform the same function on IKS channel gating to stabilize the channel open state. Here we show that similar features were observed for Kv7.1 currents expressed alone. We also find that conservation of homologous residues in helix B of other Kv7 subtypes confer similar competition of Ca2+-CaM with PIP2 binding to their proximal C-termini and suggest that PIP2-CaM interactions converge to Kv7 helix B to modulates channel activity in a Kv7 subtype-dependent manner.

New Insights into the Mechanism of Ca2+-Dependent Inactivation of NMDA Receptors

Calcium-dependent inactivation (CDI) is an important mechanism that is common to a variety of ion channels, and is particularly important to the function of the N-methyl-D-aspartic acid (NMDA) receptor (1,2). The NMDA receptor is a class of glutamate receptor that is a mediator of synaptic plasticity and has been a target for neurological drug development. In particular, spike timing-dependent plasticity is thought to be influenced by CDI (3). CDI is mediated by the interaction of calmodulin with a sequence on the intracellular C-terminal domain (C0) of the NMDA receptor 1 (NR1) subunit of the NMDA receptor (4) and by the binding of calcium to calmodulin.

Calmodulin confers calcium sensitivity to the stability of the distal intracellular assembly domain of Kv7.2 channels

Tetrameric coiled-coil structures are present in many ion channels, often adjacent to a calmodulin (CaM) binding site, although the relationship between the two is not completely understood. Here we examine the dynamic properties of the ABCD domain located in the intracellular C-terminus of tetrameric, voltage-dependent, potassium selective Kv7.2 channels. This domain encompasses the CaM binding site formed by helices A and B, followed by helix C, which is linked to the helix D coiled-coil. The data reveals that helix D stabilizes CaM binding, promoting trans-binding (CaM embracing neighboring subunits), and they suggest that the ABCD domain can be exchanged between subunits of the tetramer. Exchange is faster when mutations in AB weaken the CaM interaction. The exchange of ABCD domains is slower in the presence of Ca2+, indicating that CaM stabilization of the tetrameric assembly is enhanced when loaded with this cation. Our observations are consistent with a model that involves a dynamic mechanism of helix D assembly, which supports reciprocal allosteric coupling between the A-B module and the coiled-coil formed by the helix D. Thus, formation of the distal helix D tetramer influences CaM binding and CaM-dependent Kv7.2 properties, whereas reciprocally, CaM and Ca2+ influence the dynamic behavior of the helix D coiled-coil.

Identification of FDA-Approved Small Molecules Capable of Disrupting the Calmodulin-Adenylyl Cyclase 8 Interaction through Direct Binding to Calmodulin.

Adenylyl cyclases (AC) catalyze the formation of cyclic AMP (cAMP) from ATP and are involved in a number of disease states, making them attractive potential drug targets. AC8, in particular, has been implicated in several neurological disorders. While development of small molecule AC inhibitors has generated some chemical leads, the lack of inhibitor specificity among AC family members has limited the identification of successful drug candidates. Therefore, finding alternative novel methods to suppress AC activity are needed. Because only AC1 and AC8 are robustly stimulated by calmodulin (CaM), we set out to explore the mechanism of disrupting the AC/CaM interaction as a way to selectively inhibit AC8. Through the development and implementation of a novel biochemical high-throughput-screening paradigm, we identified six small molecules from an FDA-approved compound library that are capable of disrupting the AC8/CaM interaction. These compounds were also shown to be able disrupt formation of this complex in cells, ultimately leading to decreased AC8 activity. Interestingly, further mechanistic analysis determined that these compounds functioned by binding to CaM and blocking its interaction with AC8. While these particular compounds could inhibit CaM interaction with both AC1 and AC8, they provide significant proof of concept for inhibition of ACs through disruption of CaM binding. These compounds, as dual AC1/AC8 inhibitors, provide important tools for probing pathological conditions where AC1/AC8 activity are enhanced, such as chronic pain and ethanol consumption. Furthermore, unlike tools such as genetic deletion, these compounds can be used in a dose-dependent fashion to determine the role of AC/CaM interactions in these pathologies.

A T-type channel-calmodulin complex triggers \u03b1CaMKII activation

Calmodulin (CaM) is an important signaling molecule that regulates a vast array of cellular functions by activating second messengers involved in cell function and plasticity. Low voltage-activated calcium channels of the Cav3 family have the important role of mediating low threshold calcium influx, but were not believed to interact with CaM. We find a constitutive association between CaM and the Cav3.1 channel at rest that is lost through an activity-dependent and Cav3.1 calcium-dependent CaM dissociation. Moreover, Cav3 calcium influx is sufficient to activate αCaMKII in the cytoplasm in a manner that depends on an intact Cav3.1 C-terminus needed to support the CaM interaction. Our findings thus establish that T-type channel calcium influx invokes a novel dynamic interaction between CaM and Cav3.1 channels to trigger a signaling cascade that leads to αCaMKII activation.

CaMELS: In silico prediction of calmodulin binding proteins and their binding sites.

Due to Ca2+ -dependent binding and the sequence diversity of Calmodulin (CaM) binding proteins, identifying CaM interactions and binding sites in the wet-lab is tedious and costly. Therefore, computational methods for this purpose are crucial to the design of such wet-lab experiments. We present an algorithm suite called CaMELS (CalModulin intEraction Learning System) for predicting proteins that interact with CaM as well as their binding sites using sequence information alone. CaMELS offers state of the art accuracy for both CaM interaction and binding site prediction and can aid biologists in studying CaM binding proteins. For CaM interaction prediction, CaMELS uses protein sequence features coupled with a large-margin classifier. CaMELS models the binding site prediction problem using multiple instance machine learning with a custom optimization algorithm which allows more effective learning over imprecisely annotated CaM-binding sites during training. CaMELS has been extensively benchmarked using a variety of data sets, mutagenic studies, proteome-wide Gene Ontology enrichment analyses and protein structures. Our experiments indicate that CaMELS outperforms simple motif-based search and other existing methods for interaction and binding site prediction. We have also found that the whole sequence of a protein, rather than just its binding site, is important for predicting its interaction with CaM. Using the machine learning model in CaMELS, we have identified important features of protein sequences for CaM interaction prediction as well as characteristic amino acid sub-sequences and their relative position for identifying CaM binding sites. Python code for training and evaluating CaMELS together with a webserver implementation is available at the URL: http://faculty.pieas.edu.pk/fayyaz/software.html#camels.

Abstract 5229: High affinity interaction of K-Ras4B HVR with calmodulin

Abstract Ras proteins promote cell proliferation and survival, controlling signal transduction that involves two major pathways, such as Raf/MEK/ERK and PI3K/Akt. K-Ras4B is the most abundant oncogenic isoform. In calcium- and calmodulin (CaM)-rich environments, such as ductal tissues, it plays a critical role in adenocarcinomas promoting PI3K/Akt signaling, particularly in pancreatic, lung, and colorectal cancers. It was suggested that in adenocarcinomas with elevated calcium levels, Ca2+/CaM recruits PI3Kα through interaction with its n/cSH2 domains and sequesters K-Ras4B from the membrane, organizing a CaM/K-Ras4B/PI3Kα ternary complex. Ca2+/CaM can replace the missing receptor tyrosine kinase (RTK) signal, acting to fully activate PI3Kα. Structural data suggests that CaM uniquely binds GTP-bound K-Ras4B but not N-Ras or H-Ras isoforms. Experimental evidence points to the farnesylated hypervariable region (HVR) of K-Ras4B as a major binding domain for CaM. Here, using molecular dynamics (MD) simulations we modeled K-Ras4B HVR interaction with Ca2+/CaM. Initial contacts of K-Ras4B HVR with CaM were determined by the nuclear magnetic resonance (NMR) chemical shift perturbation(CSP) data, which guided major interacting residue pairs between the HVR and CaM. In the simulations, two different topologies of Ca2+/CaM were modeled: CaM with stretched and flexible linkers. CaM has N- and C-lobes connected by a linker, and each lobe has its own hydrophobic pocket. Thus, the hydrophobic farnesyl tail of the HVR can dock into either lobe of CaM. We observed that the HVR strongly interacts with the linker of CaM. The K-Ras4B HVR is highly polybasic with lysine-rich, while the linker region of CaM is negatively charged. The docking of the farnesyl group into the hydrophobic pocket additionally helps to stabilize the HVR/CaM interaction. This added stabilization by the farnesyl is significant in the K-Ras4B/CaM interaction even though the interaction with the Ras catalytic domain is involved. K-Ras4B has only farnesyl modification, while other isoforms bear farnesyl and palmitoyl groups. The additional lipid modification in the HVR would obstruct CaM binding, suggesting CaM's K-Ras4B-specific action. Our structural model of K-Ras4B/CaM association provides plausible clues to the PI3Kα activation involving the ternary complex. The abundant Ca2+/CaM can bind to the p85 domain of PI3Kα while binding to the K-Ras4B farnesylated HVR, releasing catalytic kinase domain autoinhibition and allosterically leading to full PI3Kα activation. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E. Citation Format: Hyunbum Jang, Avik Banerjee, Vadim Gaponenko, Ruth Nussinov. High affinity interaction of K-Ras4B HVR with calmodulin [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 5229. doi:10.1158/1538-7445.AM2017-5229

Biophysical analysis of the dynamics of calmodulin interactions with neurogranin and Ca2+ /calmodulin-dependent kinase II.

Calmodulin (CaM) functions depend on interactions with CaM‐binding proteins, regulated by Ca2+. Induced structural changes influence the affinity, kinetics, and specificities of the interactions. The dynamics of CaM interactions with neurogranin (Ng) and the CaM‐binding region of Ca2+/calmodulin‐dependent kinase II (CaMKII290−309) have been studied using biophysical methods. These proteins have opposite Ca2+ dependencies for CaM binding. Surface plasmon resonance biosensor analysis confirmed that Ca2+ and CaM interact very rapidly, and with moderate affinity ( KDSPR=3μM). Calmodulin‐CaMKII290−309 interactions were only detected in the presence of Ca2+, exhibiting fast kinetics and nanomolar affinity ( KDSPR=7.1nM). The CaM–Ng interaction had higher affinity under Ca2+‐depleted ( KDSPR=480nM,k1=3.4×105M−1s−1 and k −1 = 1.6 × 10−1s−1) than Ca2+‐saturated conditions ( KDSPR=19μM). The IQ motif of Ng (Ng27−50) had similar affinity for CaM as Ng under Ca2+‐saturated conditions ( KDSPR=14μM), but no interaction was seen under Ca2+‐depleted conditions. Microscale thermophoresis using fluorescently labeled CaM confirmed the surface plasmon resonance results qualitatively, but estimated lower affinities for the Ng ( KDMST=890nM) and CaMKII290−309( KDMST=190nM) interactions. Although CaMKII290−309 showed expected interaction characteristics, they may be different for full‐length CaMKII. The data for full‐length Ng, but not Ng27−50, agree with the current model on Ng regulation of Ca2+/CaM signaling.

Structural Consequences of Calmodulin EF Hand Mutations.

Calmodulin (CaM) is a cytosolic Ca2+-binding protein that serves as a control element for many enzymes. It consists of two globular domains, each containing two EF hand pairs capable of binding Ca2+, joined by a flexible central linker region. CaM is able to bind and activate its target proteins in the Ca2+-replete and Ca2+-deplete forms. To study the Ca2+-dependent/independent properties of binding and activation of target proteins by CaM, CaM constructs with Ca2+-binding disrupting mutations of Asp to Ala at position one of each EF hand have been used. These CaM mutant proteins are deficient in binding Ca2+ in either the N-lobe EF hands (CaM12), C-lobe EF hands (CaM34), or all four EF hands (CaM1234). To investigate potential structural changes these mutations may cause, we performed detailed NMR studies of CaM12, CaM34, and CaM1234 including determining the solution structure of CaM1234. We then investigated if these CaM mutants affected the interaction of CaM with a target protein known to interact with apoCaM by determining the solution structure of CaM34 bound to the iNOS CaM binding domain peptide. The structures provide direct structural evidence of changes that are present in these Ca2+-deficient CaM mutants and show these mutations increase the hydrophobic exposed surface and decrease the electronegative surface potential throughout each lobe of CaM. These Ca2+-deficient CaM mutants may not be a true representation of apoCaM and may not allow for native-like interactions of apoCaM with its target proteins.

Probing Conformational and Functional Substates of Calmodulin by High-Pressure FTIR

In response to intracellular Ca2+-concentration changes, the highly dynamic and flexible Ca2+-sensing protein calmodulin (CaM) interacts with more than 300 diverse target proteins that are involved in numerous signaling pathways in eukaryotic cells. This unique promiscuous target binding behavior and the underlying functional versatility of CaM is a result of its structural flexibility. CaM spans multiple conformational substates in solution providing adaptable binding surfaces for different target proteins. K-Ras4B, known as a key regulator in the ERK pathway, was identified as specific binding partner of CaM, causing the dissociation of the lipidated K-Ras4B from the plasma membrane and thus reducing its activity. However, the interaction of K-Ras4B and CaM has not been elucidated in detail until now. In order to evaluate the conformational space of CaM and shed more light on the mechanism of subsequent target recognition and protein function, identification and characterization of functionally relevant conformational substates is mandatory. Applying pressure in combination with spectroscopies such as FTIR spectroscopy enables to populate and probe otherwise transient low-lying excited conformational substates of CaM close in energy to ground state, but with a smaller partial volume. Evidence has grown that these states are functionally relevant, for instance in recognition and ligand binding events. The pressure-induced conformational changes of CaM were studied in its Ca2+-free and Ca2+-bound state and in the presence of the C-terminal K-Ras4B peptide. We show that not only Ca2+-binding, but also the presence of the target peptide has a drastic effect on the conformational dynamics of the protein.

Generating a Model for Calmodulin Induced Folding in the Disordered Regulatory Domain of Calcineurin

Calcineurin (CaN) plays an important role in the T-cell activation, cardiac system development and nervous system function. Previous studies have suggested that the regulatory domain (RD) of CaN binds Calmodulin (CaM) towards the N-terminal end. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the regulatory domain, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto inhibitory domain (AID) from the active site of CaN, activating phosphatase activity. In the absence of calcium-loaded CaM, the RD is at least partially disordered, and binding of CaM induces folding in the RD. Previous studies have shown that an α-helical structure forms in the N-terminal half of the RD, but organization may occur in the C-terminal half as well. Here, we are interested in the structural transition of the full length RD as it binds to CaM. Using nuclear magnetic resonance (NMR) spectroscopy, we have successfully assigned >85% of the 15N, 13Cα, 13Cβ and HN chemical shifts of the unbound, regulatory domain of CaN. While the protein is disordered, secondary chemical shifts indicate that some regions possess α-helical propensity, even in the unbound state. Our study of the CaM and CaN interaction supports the formation of a distal helix in the region between the AID and calmodulin-binding region. Heat capacity changes upon binding predict that 54 residues fold when CaM binds to CaN, consistent with the formation of this distal helix. Paramagnetic relaxation enhancement (PRE) studies of this interaction suggest a potential binding mode where the distal helix binds to CaM near residues I9-A15. Mutagenesis in the distal helix disrupts PREs, further supporting this hypothesis. Together, these data suggest that the interactions between CaM and the distal helix of CaN can be important in regulation of phosphatase activity.

EPR Structural Dynamics of Calmodulin-RyR Peptide Complex

We have used a combination of site-directed spin labeling and EPR spectroscopy to probe the interaction of calmodulin (CaM) with a peptide (RyRp) corresponding to the CaM-binding site on the ryanodine receptor, the muscle calcium release channel. A TOAC spin label, which is bifunctionally and stereospecifically coupled to the alpha-carbon and directly reports peptide backbone dynamics, was used to label RyRp at positions 5, 12, 18, or 25. CW-EPR showed that RyRp alone exhibits a gradient of rotational dynamics (mobility), with the N-terminal region having greater mobility than the C-terminal region. Addition of CaM restricted mobility at all four positions, while preserving the mobility gradient. In the presence of saturating Ca, the immobilization of TOAC-RyRp by CaM is further increased. A bifunctional spin label (BSL) attached stereospecifically at Cys sites i and i+4 was used to label CaM at positions 34 and 38 in the N-lobe and 106 and 110 in the C-lobe. Interspin distances measured by DEER indicated that CaM, in the absence of peptide and Ca, exists in a closed conformation, while Ca induces a shift in equilibrium toward two additional conformations, one more open and another more closed (“compact”). Addition of RyRp caused the two lobes of CaM to collapse toward each other (compact conformation), while exhibiting a broad distribution, indicating high flexibility. Removal of Ca from the complex caused increased distance between the spin labels, indicating greater lobe separation. Distances measured between BSL (on CaM) and TOAC (on RyRp) confirmed the compact but flexible conformation of the complex. These results provide insight into the structural dynamics of the Ca-dependent regulation of RyR by CaM.

The Interplay between Calmodulin and Membrane Interactions with the Pleckstrin Homology Domain of Akt

The Akt protein, a serine/threonine kinase, plays important roles in cell survival, apoptosis, and oncogenes. Akt is translocated to the plasma membrane for activation. Akt-membrane binding is mediated by direct interactions between its pleckstrin homology domain (PHD) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). It has been shown that Akt activation in breast cancer cells is modulated by calmodulin (CaM). However, the molecular mechanism of the interplay between CaM and membrane binding is not established. Here, we employed nuclear magnetic resonance (NMR) and biochemical and biophysical techniques to characterize how PI(3,4,5)P3, CaM, and membrane mimetics (nanodisc) bind to Akt(PHD). We show that PI(3,4,5)P3 binding to Akt(PHD) displaces the C-terminal lobe of CaM but not the weakly binding N-terminal lobe. However, binding of a PI(3,4,5)P3-embedded membrane nanodisc to Akt(PHD) with a 103-fold tighter affinity than PI(3,4,5)P3 is able to completely displace CaM. We also show that Akt(PHD) binds to both layers of the nanodisc, indicating proper incorporation of PI(3,4,5)P3 on the nanodisc surface. No detectable binding has been observed between Akt(PHD) and PI(3,4,5)P3-free nanodiscs, demonstrating that PI(3,4,5)P3 is required for membrane binding, CaM displacement, and Akt activation. Using pancreatic cancer cells, we demonstrate that inhibition of Akt-CaM binding attenuated Akt activation. Our findings support a model by which CaM binds to Akt to facilitate its translocation to the membrane. Elucidation of the molecular details of the interplay between membrane and CaM binding to Akt may help in the development of potential targets to control the pathophysiological processes of cell survival.

Calcium-dependent binding of Myc to calmodulin.

The bHLH-LZ (basic region/helix-loop-helix/leucine zipper) oncoprotein Myc and the bHLH-LZ protein Max form a binary transcription factor complex controlling fundamental cellular processes. Deregulated Myc expression leads to neoplastic transformation and is a hallmark of most human cancers. The dynamics of Myc transcription factor activity are post-translationally coordinated by defined protein-protein interactions. Here, we present evidence for a second messenger controlled physical interaction between the Ca2+ sensor calmodulin (CaM) and all Myc variants (v-Myc, c-Myc, N-Myc, and L-Myc). The predominantly cytoplasmic Myc:CaM interaction is Ca2+-dependent, and the binding site maps to the conserved bHLH domain of Myc. Ca2+-loaded CaM binds the monomeric and intrinsically disordered Myc protein with high affinity, whereas Myc:Max heterodimers show less, and Max homodimers no affinity for CaM. NMR spectroscopic analyses using alternating mixtures of 15N-labeled and unlabeled preparations of CaM and a monomeric Myc fragment containing the bHLH-LZ domain corroborate the biochemical results on the Myc:CaM interaction and confirm the interaction site mapping. In electrophoretic mobility shift assays, addition of CaM does not affect high-affinity DNA-binding of Myc:Max heterodimers. However, cell-based reporter analyses and cell transformation assays suggest that increasing CaM levels enhance Myc transcriptional and oncogenic activities. Our results point to a possible involvement of Ca2+ sensing CaM in the fine-tuning of Myc function.

Characterization of the interactions between calmodulin and death receptor 5 in triple-negative and estrogen receptor-positive breast cancer cells. AN INTEGRATED EXPERIMENTAL AND COMPUTATIONAL STUDY.

VOLUME 291 (2016) PAGES 12862–12870 Thomas Schmid was inadvertently omitted as an author. The author line should appear as shown. Thomas Schmid contributed to the computational studies and was supported by the U. S. Department of State through a Fulbright scholarship. His primary affiliation is with the Department of Biomedical Engineering at the University of Alabama at Birmingham, Birmingham, Alabama 35294, and his secondary affiliation is with the Department of Computer Engineering, Universität Leipzig, Augustusplatz 10, D-04109 Leipzig, Germany. Additionally, Thomas Schmid, Qinghua Zeng, and Hong Wang contributed equally to this study.

Current view on regulation of voltage-gated sodium channels by calcium and auxiliary proteins.

In cardiac and skeletal myocytes, and in most neurons, the opening of voltage-gated Na(+) channels (NaV channels) triggers action potentials, a process that is regulated via the interactions of the channels' intercellular C-termini with auxiliary proteins and/or Ca(2+) . The molecular and structural details for how Ca(2+) and/or auxiliary proteins modulate NaV channel function, however, have eluded a concise mechanistic explanation and details have been shrouded for the last decade behind controversy about whether Ca(2+) acts directly upon the NaV channel or through interacting proteins, such as the Ca(2+) binding protein calmodulin (CaM). Here, we review recent advances in defining the structure of NaV intracellular C-termini and associated proteins such as CaM or fibroblast growth factor homologous factors (FHFs) to reveal new insights into how Ca(2+) affects NaV function, and how altered Ca(2+) -dependent or FHF-mediated regulation of NaV channels is perturbed in various disease states through mutations that disrupt CaM or FHF interaction.

Characterization of the Interactions between Calmodulin and Death Receptor 5 in Triple-negative and Estrogen Receptor-positive Breast Cancer Cells: AN INTEGRATED EXPERIMENTAL AND COMPUTATIONAL STUDY

Activation of death receptor-5 (DR5) leads to the formation of death inducing signaling complex (DISC) for apoptotic signaling. Targeting DR5 to induce breast cancer apoptosis is a promising strategy to circumvent drug resistance and present a target for breast cancer treatment. Calmodulin (CaM) has been shown to regulate DR5-mediated apoptotic signaling, however, its mechanism remains unknown. In this study, we characterized CaM and DR5 interactions in breast cancer cells with integrated experimental and computational approaches. Results show that CaM directly binds to DR5 in a calcium dependent manner in breast cancer cells. The direct interaction of CaM with DR5 is localized at DR5 death domain. We have predicted and verified the CaM-binding site in DR5 being (354)WEPLMRKLGL(363) that is located at the α2 helix and the loop between α2 helix and α3 helix of DR5 DD. The residues of Trp-354, Arg-359, Glu-355, Leu-363, and Glu-367 in DR5 death domain that are important for DR5 recruitment of FADD and caspase-8 for DISC formation to signal apoptosis also play an important role for CaM-DR5 binding. The changed electrostatic potential distribution in the CaM-binding site in DR5 DD by the point mutations of W354A, E355K, R359A, L363N, or E367K in DR5 DD could directly contribute to the experimentally observed decreased CaM-DR5 binding by the point mutations of the key residues in DR5 DD. Results from this study provide a key step for the further investigation of the role of CaM-DR5 binding in DR5-mediated DISC formation for apoptosis in breast cancer cells.